Inhibitors of hedgehog signaling pathways, compositions and uses related thereto

ABSTRACT

The present invention makes availables assays and reagents inhibiting paracrine and/or autocrine signals produced by a hedgehog protein comprising contacting a cell sensitive to the hedgehog protein with a steroidal alkaloid, or other small molecule, in a sufficient amount to reduce the sensitivity of the cell to the hedgehog protein.

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional application No.60/081,186, filed Apr. 9, 1998.

BACKGROUND OF THE INVENTION

Pattern formation is the activity by which embryonic cells form orderedspatial arrangements of differentiated tissues. The physical complexityof higher organisms arises during embryogenesis through the interplay ofcell-intrinsic lineage and cell-extrinsic signaling. Inductiveinteractions are essential to embryonic patterning in vertebratedevelopment from the earliest establishment of the body plan, to thepatterning of the organ systems, to the generation of diverse cell typesduring tissue differentiation (Davidson, E., (1990) Development 108:365-389; Gurdon, J. B., (1992) Cell 68: 185-199; Jessell, T. M. et al.,(1992) Cell 68: 257-270). The effects of developmental cell interactionsare varied. Typically, responding cells are diverted from one route ofcell differentiation to another by inducing cells that differ from boththe uninduced and induced states of the responding cells (inductions).Sometimes cells induce their neighbors to differentiate like themselves(homeogenetic induction); in other cases a cell inhibits its neighborsfrom differentiating like itself. Cell interactions in early developmentmay be sequential, such that an initial induction between two cell typesleads to a progressive amplification of diversity. Moreover, inductiveinteractions occur not only in embryos, but in adult cells as well, andcan act to establish and maintain morphogenetic patterns as well asinduce differentiation (J. B. Gurdon (1992) Cell 68:185-199).

Members of the Hedgehog family of signaling molecules mediate manyimportant short- and long-range patterning processes during invertebrateand vertebrate development. In the fly a single hedgehog gene regulatessegmental and imaginal disc patterning. In contrast, in vertebrates ahedgehog gene family is involved in the control of left-right asymmetry,polarity in the CNS, somites and limb, organogenesis, chondrogenesis andspermatogenesis.

The first hedgehog gene was identified by a genetic screen in thefruitfly Drosophila melanogaster (Nüsslein-Volhard, C. and Wieschaus, E.(1980) Nature 287, 795-801). This screen identified a number ofmutations affecting embryonic and larval development. In 1992 and 1993,the molecular nature of the Drosophila hedgehog (hh) gene was reported(C. F., Lee et al. (1992) Cell 71, 33-50), and since then, severalhedgehog homologues have been isolated from various vertebrate species.While only one hedgehog gene has been found in Drosophila and otherinvertebrates, multiple Hedgehog genes are present in vertebrates.

The various Hedgehog proteins consist of a signal peptide, a highlyconserved N-terminal region, and a more divergent C-terminal domain. Inaddition to signal sequence cleavage in the secretory pathway (Lee, J.J. et al. (1992) Cell 71:33-50; Tabata, T. et al. (1992) Genes Dev.2635-2645: Chang, D. E. et al. (1994) Development 120:3339-3353),Hedgehog precursor proteins undergo an internal autoproteolytic cleavagewhich depends on conserved sequences in the C-terminal portion (Lee etal. (1994) Science 266:1528-1537; Porter et al. (1995) Nature374:363-366). This autocleavage leads to a 19 kD N-terminal peptide anda C-terminal peptide of 26-28 kD (Lee et al. (1992) supra; Tabata et al.(1992) supra; Chang et al. (1994) supra; Lee et al. (1994) supra;Bumcrot, D. A., et al (1995) Mol. Cell. Biol. 15:2294-2303; Porter etal. (1995) supra; Ekker, S. C. et al. (1995) Curr. Biol. 5:944-955; Lai,C. J. et al. (1995) Development 121:2349-2360). The N-terminal peptidestays tightly associated with the surface of cells in which it wassynthesized, while the C-terminal peptide is freely diffusible both invitro and in vivo (Porter et al. (1995) Nature 374:363; Lee et al.(1994) supra; Bumcrot et al. (1995) supra; Mart', E. et al. (1995)Development 121:2537-2547; Roelink, H. et al. (1995) Cell 81:445-455).Interestingly, cell surface retention of the N-terminal peptide isdependent on autocleavage, as a truncated form of HH encoded by an RNAwhich terminates precisely at the normal position of internal cleavageis diffusible in vitro (Porter et al. (1995) supra) and in vivo (Porter,J. A. et al. (1996) Cell 86, 21-34). Biochemical studies have shown thatthe autoproteolytic cleavage of the HH precursor protein proceedsthrough an internal thioester intermediate which subsequently is cleavedin a nucleophilic substitution. It is likely that the nucleophile is asmall lipophilic molecule which becomes covalently bound to theC-terminal end of the N-peptide (Porter et al. (1996) supra), tetheringit to the cell surface. The biological implications are profound. As aresult of the tethering, a high local concentration of N-terminalHedgehog peptide is generated on the surface of the Hedgehog producingcells. It is this N-terminal peptide which is both necessary andsufficient for short and long range Hedgehog signaling activities inDrosophila and vertebrates (Porter et al. (1995) supra; Ekker et al.(1995) supra; Lai et al (1995) supra; Roelink, H. et al. (1995) Cell81:445-455; Porter et al. (1996) supra; Fietz, M. J. et al. (1995) Curr.Biol. 5:643-651; Fan, C. -M. et al. (1995) Cell 81:457-465; Mart', E.,et al. (1995) Nature 375:322-325; Lopez-Martinez et al. (1995) Curr.Biol 5:791-795: Ekker, S. C. et al. (1995) Development 121:2337-2347;Forbes, A. J. et al.(1996) Development 122:1125-1135).

HH has been implicated in short- and longe range patterning processes atvarious sites during Drosophila development. In the establishment ofsegment polarity in early embryos, it has short range effects whichappear to be directly mediated, while in the patterning of the imaginaldiscs, it induces long range effects via the induction of secondarysignals.

In vertebrates, several hedgehog genes have been cloned in the past fewyears. Of these genes, Shh has received most of the experimentalattention, as it is expressed in different organizing centers which arethe sources of signals that pattern neighbouring tissues. Recentevidence indicates that Shh is involved in these interactions.

The expression of Shh starts shortly after the onset of gastrulation inthe presumptive midline mesoderm, the node in the mouse (Chang et al.(1994) supra; Echelard, Y. et al. (1993) Cell 75:1417-1430), the rat(Roelink, H. et al. (1994) Cell 76:761-775) and the chick (Riddle, R. D.et al. (1993) Cell 75:1401-1416), and the shield in the zebrafish (Ekkeret al. (1995) supra; Krauss, S. et al. (993) Cell 75:1431-1444). Inchick embyros, the Shh expression pattern in the node develops aleft-right asymmetry, which appears to be responsible for the left-rightsitus of the heart (Levin, M. et al. (1995) Cell 82:803-814).

In the CNS, Shh from the notochord and the floorplate appears to induceventral cell fates. When ectopically expressed, Shh leads to aventralization of large regions of the mid- and hindbrain in mouse(Echelard et al. (1993) supra; Goodrich, L. V. et al. (1996) Genes Dev.10:301-312), Xenopus (Roelink, H. et al. (1994) supra; Ruiz i Altaba, A.et al. (1995) Mol. Cell. Neurosci. 6:106-121), and zebrafish (Ekker etal. (1995) supra; Krauss et al. (1993) supra; Hammerschmidt, M., et al.(1996) Genes Dev. 10:647-658). In explants of intermediate neuroectodermat spinal cord levels, Shh protein induces floorplate and motor neurondevelopment with distinct concentration thresholds, floor plate at highand motor neurons at lower concentrations (Roelink et al. (1995) supra;Mart' et al. (1995) supra; Tanabe, Y. et al. (1995) Curr. Biol.5:651-658). Moreover, antibody blocking suggests that Shh produced bythe notochord is required for notochord mediated induction of motorneuron fates (Mart' et al. (1995) supra). Thus, high concentration ofShh on the surface of Shh-producing midline cells appears to account forthe contact-mediated induction of Doorplate observed in vitro (Placzek,M. et al. (1993) Development 117:205-218), and the midline positioningof the floorplate immediately above the notochord in vivo. Lowerconcentrations of Shh released from the notochord and the floorplatepresumably induce motor neurons at more distant ventrolateral regions ina process that has been shown to be contact-independent in vitro(Yamada, T. et al. (1993) Cell 73:673-686). In explants taken atmidbrain and forebrain levels, Shh also induces the appropriateventrolateral neuronal cell types, dopaminergic (Heynes, M. et al.(1995) Neuron 15:35-44; Wang, M. Z. et al. (1995) Nature Med.1:1184-1188) and cholinergic (Ericson, J. et al. (1995) Cell 81:747-756)precursors, respectively, indicating that Shh is a common inducer ofventral specification over the entire length of the CNS. Theseobservations raise a question as to how the differential response to Shhis regulated at particular anteroposterior positions.

Shh from the midline also patterns the paraxial regions of thevertebrate embryo, the somites in the trunk (Fan et al. (1995) supra)and the head mesenchyme rostral of the somites (Hammerschmidt et al.(1996) supra). In chick and mouse paraxial mesoderm explants, Shhpromotes the expression of sclerotome specific markers like Pax1 andTwist, at the expense of the dermamyotomal marker Pax3. Moreover, filterbarrier experiments suggest that Shh mediates the induction of thesclerotome directly rather than by activation of a secondary signalingmechanism (Fan, C. -M. and Tessier-Lavigne, M. (1994) Cell 79,1175-1186).

Shh also induces myotomal gene expression (Hammerschmidt et al. (1996)supra; Johnson, R. L. et al. (1994) Cell 79:1165-1173; Münsterberg, A.E. et al. (1995) Genes Dev. 9:2911-2922; Weinberg, E. S. et al. (1996)Development 122:271-280), although recent experiments indicate thatmembers of the WNT family, vertebrate homologues of Drosophila wingless,are required in concert (Münsterberg et al. (1995) supra). Puzzlingly,myotomal induction in chick requires higher Shh concentrations than theinduction of sclerotomal markers (Münsterberg et al. (1995) supra),although the sclerotome originates from somitic cells positioned muchcloser to the notochord. Similar results were obtained in the zebrafish,where high concentrations of Hedgehog induce myotomal and represssclerotomal marker gene expression (Hammerschmidt et al. (1996) supra).In contrast to amniotes, however, these observations are consistent withthe architecture of the fish embryo, as here, the myotome is thepredominant and more axial component of the somites. Thus, modulation ofShh signaling and the acquisition of new signaling factors may havemodified the somite structure during vertebrate evolution.

In the vertebrate limb buds, a subset of posterior mesenchymal cells,the “Zone of polarizing activity” (ZPA), regulates anteroposterior digitidentity (reviewed in Honig, L. S. (1981) Nature 291:72-73). Ectopicexpression of Shh or application of beads soaked in Shh peptide mimicsthe affect of anterior ZPA grafts, generating a mirror image duplicationof digits (Chang et al. (1994) supra; Lopez-Martinez et al. (1995)supra; Riddle et al. (1993) supra) (FIG. 2g). Thus, digit identityappears to depend primarily on Shh concentration, although it ispossible that other signals may relay this information over thesubstantial distances that appear to be required for AP patterning(100-150 μm). Similar to the interaction of HH and DPP in the Drosophilaimaginal discs, Shh in the vertebrate limb bud activates the expressionof Bmp2 (Francis, P. H. et al. (1994) Development 120:209-218), a dpphomologue. However, unlike DPP in Drosophila, BMP2 fails to mimic thepolarizing effect of Shh upon ectopic application in the chick limb bud(Francis et al. (1994) supra). In addition to anteroposteriorpatterning, Shh also appears to be involved in the regulation of theproximodistal outgrowth of the limbs by inducing the synthesis of thefibroblast growth factor FGF4 in the posterior apical ectodermal ridge(Laufer, E. et al. (1994) Cell 79:993-1003; Niswander, L. et al. (1994)Nature 371:609-612).

The close relationship between Hedgehog proteins and BMPs is likely tohave been conserved at many, but probably not all sites of vertebrateHedgehog expression. For example, in the chick hindgut, Shh has beenshown to induce the expression of Bmp4, another vertebrate dpp homologue(Roberts. D. J. et al. (1995) Development 121:3163-3174). Furthermore,Shh and Bmp2,4, or 6 show a striking correlation in their expression inepithelial and mesenchymal cells of the stomach, the urogential system,the lung, the tooth buds and the hair follicles (Bitgood, M. J. andMcMahon, A. P. (1995) Dev. Biol. 172:126-138). Further, Ihh, one of thetwo other mouse Hedgehog genes, is expressed adjacent to Bmp expressingcells in the gut and developing cartilage (Bitgood and McMahon (1995)supra).

Recent evidence suggests a model in which Ihh plays a crucial role inthe regulation of chondrogenic development (Roberts et al. (1995)supra). During cartilage formation, chondrocytes proceed from aproliferating state via an intermediate, prehypertrophic state todifferentiated hypertrophic chondrocytes. Ihh is expressed in theprehypertrophic chondrocytes and initiates a signaling cascade thatleads to the blockage of chondrocyte differentiation. Its direct targetis the perichondrium around the Ihh expression domain, which responds bythe expression of Gli and Patched (Ptc), conserved transcriptionaltargets of Hedgehog signals (see below). Most likely, this leads tosecondary signaling resulting in the synthesis of parathyroidhormone-related protein (PTHrP) in the periarticular perichondrium.PTHrP itself signals back to the prehypertrophic chondrocytes, blockingtheir further differentiation. At the same time, PTHrP repressesexpression of Ihh, thereby forming a negative feedback loop thatmodulates the rate of chondrocyte differentiation.

SUMMARY OF THE INVENTION

The present invention makes availables assays and reagents inhibitingparacrine and/or autocrine signals produced by a hedgehog proteincomprising contacting a cell sensitive to the hedgehog protein with asteroidal alkaloid, or other small molecule, in a sufficient amount toreduce the sensitivity of the cell to the hedgehog protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Structures of the synthetic compounds AY 9944 and triparanol, ofthe plant steriodal alkaloids jervine, cyclopamine and tomatidine, andof cholesterol.

FIG. 2. Holoprosencephaly induced in chick embryos exposed to jervine(4). (A) SEM of external facial features of an untreated embryo. (B, C,D and E) Embryos exposed to 10 μ_M jervine with variable loss of midlinetissue and resulting fusion of the paired, lateral olfactory processes(olf), optic vesicles (Opt), and maxillary (Mx) and mandibular (Mn)processes. A complete fusion of the optic vesicles (E) lead to truecyclopia.

FIG. 3. Synthetic and plant derived teratogens block endogenous Shhsignaling in explanted chick tissues (41). (A) Midline tissue wasremoved from stage 9-10 chick embryos at a level just rostral toHensen's node (white dashed line), and further dissected (black dashedlines) to yield an explant containing an endogenous source of Shh signal(notochord) and a responsive tissue (neural plate ectoderm). After twodays of culture in a collagen gel matrix, the neural ectoderm expressesmarkers of floor plate cells (HNF3β, rhodamine) and motor neurons(Isl-1, FITC) in untreated control explants (B) and explants culturedwith the non-teratogenic alkaloid tomatidine (50 μM, C). Intermediadoses of the teratogenic compounds AY 9944 (0.5 μM, D), triparanol (0.25μM, E), jervine (0.5 μM, F) and cyclopamine (0.25 μM, G) block inductionof HNF3β, which requires a high level of Shh pathway activation, whilepermitting induction of Isl-1, which requires a lower level of Shhpathway activation (see text). Higher doses of the teratogenic compoundsAY 9944 (4.0 μM, H), triparanol (1.0 μM, I), jervine (4.0 μM, J) andcyclopamine (1.0 μM, K) and fully inhibit HNF3β and Isl-1 induction.

FIG. 4. Teratogenic compounds do not inhibit Shh autoprocessing in vivo(47). Stably transfected HK293 cells expressing Shh protein underecdysone-inducible control (lanes 1, 2, 3) were treated with jervine(lanes 4, 5) cyclopamine (lanes 6, 7), tomatidine (lanes 10, 11), AY9944 (lanes 12, 13) or triparanol (lanes 14, 15) and cell lysates wereimmunoblotted to assess the efficiency of autoprocessing. As seen in theuntreated control (lane 3). Shh in treated cells is efficientlyprocessed with little or no detectable accumulation of precursor protein(M_(r)45 kD). The processed amino-terminal product (Shh-N_(p)) is cellassociated and migrates faster than Shh-N protein from the media ofcultured cells transfected with a construct carrying an open readingframe truncated after Gly₁₉₈ (lane 8; Shh-N_(p) and Shh-N both loaded inlanes 9 and 17). This faster migration and the lack of detectableprotein in the culture medium (not shown) indicate that Shh-N_(p) fromtreated cells likely carries a sterol adduct. The slower migratingspecies resulting from tomatidine treatment is ˜1.9 kD larger,suggestive of a minor inhibition of signal sequence cleavage (seeasterisk; lanes 10, 11). Immunoblotted actin for each lane is shown as aloading control.

FIG. 5. Plant steriodal alkaloids do not inhibit or participate in Hhautoprocessing in vitro (5). (A) Coomassie blue-stainedSDS-polyacrylamide gel showing in vitro autocleavage reactions of thebaterically expressed His₆Hh-C protein (˜29 kD) incubated for 3 hours at30° C. with no sterol additions (lane 1) or 12 μM cholesterol tostimulate the autoprocessing reaction and generate a ˜25 kD Hh-C product(lanes 2-27 and a ˜5 kD NH₂-terminal product (not resolved on this gel).The addition of jervine (lanes 3-6), cyclopamine (lanes 8-11) andtomatidine (lanes 13-16) does not interfere with autoprocessing, evenwhen added in 27-fold excess to cholesterol (lanes 6, 11 and 16). (B)Coomassie blue-stained SDS-polyacrylamide gel showing that the His₆Hh-Cautocleavage reaction does not proceed when carried out in the absenceof sterol (lane 1), or in the presence of jervine (lanes 2-5),cyclopamine (lanes 6-9) and tomatidine (lanes 10-13), even at 324 μMconcentrations of these steriodal alkaloid (lanes 5, 9 and 13). (C)Coomassie blue-stained SDS0 polyacrylamide gel of His₆Hh-C autocleavagereactions carried out in the absence of sterols (lane 1), with 50 mMdithiothreitol (lane 2), 12 μM cholesterol (lane 3) 12 μM7dehydrocholesterol (lane 4) 12 μM desmosterol (lane 5), 12 μMmuristerone (lanes 9, 10). The 27-carbon cholesterol precursors (lanes4-6) stimulate His₆Hh-C autocleavage reactions carried out in theabsence of sterols (lane 1), with 50 mM dithiothreitol (lane 2), 12 μMcholesterol (lane 3) 12 μM 7 dehydrocholesterol (lane 4) 12 μMlathosterol (lane 6), 12 and 350 μM lanosterol (lanes 7, 8) and 12 and350 μM muristerone (lanes 9, 10). The 27-carbon cholesterol precursors(lanes 4-6) stimulate His₆Hh-C autoprocessing as efficiently ascholesterol (lane 3). The amino-terminal product migrates as a ˜7 kDspecies (lane 2) when generated in the presence of 50 mM dithiothreitoland as a ˜5 kD species (lanes 3-6) with a sterol adduct. Lanosterol(lanes 7 and 8) and muristerone (lanes 9 and 10) do not stimulateautoprocessing above background (lane 1).

FIG. 6. Teratogenic compounds inhibit neural ectoderm response toexogenous Shh-N protein (41). (A) Intermediate neural plate ectoderm,free of notochord and other tissues, was dissected as shown (dashedlines) from stage 9-10 chick embryos at a level just rostral to Hensen'snode (see FIG. 3A). (B) Explanted intermediate neural plate tissuecultured in a collagen gel matrix for 20 hours expresses the dorsalmarker Pax7 (FITC) and not the floor plate marker HNF3β (Rhodamine). (C)Addition of recombinant, purified Shh-N at 2 nM suppresses Pax7expression. (D) Markers of motor neuron (Isl-1, FITC) and floor platecell (HNF3β, rhodamine) fates are induced upon explant culture for 40hours in the presence of 6.25 nM Shh-N. (E) At 25 nM Shh-N, HNF3βexpression expands at the expense of Isl-1 expression, which is lost.The repression of Pax7 expression by 2 nM Shh-N is inhibited by (F) 0.5μM AY 9944, (G) 0.25 μM triparanol, (H) 0.125 μM jervine and (1) 0.0625μM cyclopamine, but not by (J) 50 μM tomatidine. Induction of HNF3β isblocked while induction of Isl-1 at 25 NM Shh-N is maintained orexpanded at intermediate levels of AY 9949 (1.0 μM, K), triparanol (0.25μM, L), jervine (0.25 μM, M), and cyclopamine (0.125 μM, N). Tomatidineat 25 nM displays a slight inhibitory effect with decrease in HNF3βexpression and an increase in the number Isl-1 expressing cells. HNF3βand Isl-1 induction are completely blocked at 2-fold higher doses ofinhibitors AY 9944 (2.0 μM, P), triparanol (0.5 μM, Q), jervine (0.5,μM, R) and cyclopamine (0.25 μM, S). Tomatidine at 50 μM (T) markedlyreduces HNF3β induction and enhances Isl1-1 induction. Note that foreach teratogenic compound the concentrations required to block completethe response to 2 nM Shh-N (F-I) are lower than those required to blockcompletely the response to 25 nM Shh-N (P-S). Also note that theresponse to 25 nM Shh-N is only partially inhibited (K-N) atconcentrations of teratogen 2 fold lower than those required to blockthis response completely. See text for further comment.

FIG. 7. Jervine does not inhibit neural ectoderm response to BMP7 (41).(A) Ventral neural plate ectoderm was dissected as shown (dashed lines)from stage 9-10 chick embryos at a level just rostral to Hensen's node(see FIG. 3A). (B) Ventral neural plate explants cultured for 24 hoursin a collagen gel matrix do not give rise to any migratory cells thatcan be visualized by immunostaining for the HNK-1 antigen. (C) Additionof 100 ng/ml BMP7 induces formation of numerous HNK-1 positive cellsthat migrate out from the explant (borders outlined by white dashedline). (D) Induction of migratory HNK-1 positive cells by 100 ng/ml BMP7is not inhibited by the presence of 10 μM jervine, nor by addition ofthe other plant-derived compounds (10 μM cyclopamine. 50 μM tomatidine;data not shown).

DETAILED DESCRIPTION OF THE INVENTION

Hedgehog (hedgehog) proteins comprise a family of secreted signalingmolecules essential for patterning a variety of structures in animalembryo genesis, and play a role in regulating cell proliferation andspecifying cell identity in diverse systems in adults.

During biosynthesis, hedgehog undergoes an autocleavage reaction,mediated by its carboxyl-terminal domain, that produces a lipid-modifiedamino-terminal fragment responsible for all known hedgehog signalingactivity. In addition to peptide bond cleavage, hedgehog autoprocessingcauses the covalent attachment of a lipophilic adduct to theCOOH-terminus of N-terminal hedgehog fragment. This modification iscritical for the spatially restricted tissue localization of thehedgehog signal; in its absence, the signaling domain exerts aninappropriate influence beyond its site of expression. It has recentlybeen reported, Porter et al. (1996) Science 274:255, that cholesterol isthe lipophilic moiety covalently attached to the amino-terminalsignaling domain during autoprocessing and that the carboxyl-terminaldomain acts as an intramolecular cholesterol transferase. This use ofcholesterol to modify the hedgehog signaling proteins is consistent withsome of the effects that perturbed cholesterol biosynthesis can have onanimal development. See also Volhard et al. (1998) Nature 287 795;Mohleret al. (1988) Genetics 120:1061; Lee et al. (1992) Cell 71:33;Tabata et al. (1992) Genes Dev 6:2635; Tashiro et al. (1993) Genel24:183; P. W. Ingham, Nature 366:560 (1993); Mohler et al. Development115:957 (1992); Ma et al. Cell 75:927 (1993); Heberlein et al. ibid,p.913; Echelard et al., Ibid, p. 1417; Riddle et al. ibid., p. 1401;Krauss et al., ibid., p. 1431; Roelink et al., ibid 76, 761 (1994);Chang et al., Development 120:3339 (1994); Basler et al. Nature 368:208(1994); Tabata et al. Cell 76:89 (1994); Heemskerk et al., ibid., p.449; Fan et al., ibid. 79:1175 (1994); Johnson et al., ibid., p. 1165;Hynes et al., Neuron 15:35 (1995); Ekker et al., Development 121:2337(1995); Macdonald et al., ibid. p. 3267; Ekker et al., Curr. Biol 5:944(1995); Lai et al. Development 121, 2349 (1995); Ericson et al., Cell81:747 (1995). Chiang et al., Nature 83:407 (1996); Bitgood et al. CurrBiol. 6:298 (1996); Vortkamp et al., Science 273:613 (1996); Lee et al.,ibid. 266:1528 (1994); Porter et al., Nature 374: 363 (1995); Porter etal. Cell 86:21 (1996).

I. Overview

The present invention relates to the discovery that signal transductionpathways dependent on hedgehog proteins can be inhibited, at least inpart, by compounds which disrupt the cholesterol modification ofhedgehog proteins and/or which inhibit the bioactivity of hedgehogproteins. In particular, Applicants believe that they are the first todemonstrate that a small molecule, e.g., having a molecular weight lessthan 2500 amu, is capable of inhibiting at least some of the biologicalactivities of hedgehog proteins.

One aspect of the present invention relates to the use of steroidalalkaloids, and analogs thereof, to interfere with paracrine and/orautocrine signals produced by the hedgehog proteins, particularlycholesterol-modified (CM) forms of the proteins. As set out in moredetail below, we have observed that members of the steroidal alkaloidclass of compounds, such as the Veratrum-derived compound jervine,disrupt cholesterol-mediated activities of the hedgehog proteins.

While not wishing to bound by any particular theory, the ability ofjervine and other steroidal alkaloids to inhibit hedgehog signalling maybe due to the ability of such molecules to interact with the sterolsensing domain(s) of the hedgehog receptor, patched, or at least tointefere with the ability of a hedgehog protein, e.g., acholesterol-modified protein, to interact with its receptor, or othermolecules associated with the receptor, or proteins otherwise involvedin hedgehog-mediated signal transduction.

Alternatively, or in addition to such a mechanism of action, the effectsof jervine on hedgehog signaling could be the result of perturbations ofcholesterol homeostasis which affect cholesterol-mediated autoprocessingof the hedgehog protein and or the activity or stability of protein. Inparticular, as described in the appended examples, Jervine and other ofthe steroidal alkaloids are so-called “class 2” inhibitors ofcholesterol biosynthesis, that is they inhibit the inward flux ofsterols. As described by Lange and Steck (1994) J Biol Chem 269:29371-4, these inhibitors immediately inhibit plasma membranecholesterol esterification and progressively induce3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and sterolbiosynthesis. The change in the relative cholesterol levels can effect,e.g., the activity and/or stability of ptc. According to the presentinvention, the subject methods may be carried out utilizing other agentswhich perturb cholesterol homeostasis in a manner similar to jervine.

It is, therefore, specifically contemplated that other small molecules,steroidal and non-steroidal in structure, which similarly intefere withcholesterol dependent aspects of ptc activity will likewise be capableof disrupting hedgehog-mediated signals. In preferred embodiments, thesubject inhibitors are organic molecules having a molecular weight lessthan 2500 amu, more preferably less than 1500 amu, and even morepreferagly less than 750 amu, and are capable of inhibiting at leastsome of the biological activities of hedgehog proteins.

Thus, the methods of the present include the use of steroidal alkaloids,and other small molecules, which antagonize hedgehog signalling in theregulation of repair and/or functional performance of a wide range ofcells, tissues and organs, and have therapeutic and cosmeticapplications ranging from regulation of neural tissues, bone andcartilage formation and repair, regulation of spermatogenesis,regulation of smooth muscle, regulation of lung, liver and other organsarising from the primative gut, regulation of hematopoietic function,regulation of skin and hair growth, etc. Accordingly, the methods andcompositions of the present invention include the use of the subjectinhibitors for all such uses as antagonists of hedgehog proteins may beimplicated. Moreover, the subject methods can be performed on cellswhich are provided in culture (in vitro), or on cells in a whole animal(in vivo). See, for example, PCT publications WO 95/18856 and WO96/17924 (the specifications of which are expressly incorporated byreference herein).

In one aspect, the present invention provides pharmaceuticalpreparations comprising, as an active ingredient, an inhibitor ofcholesterol-mediated hedgehog bioactivity, such as described herein.

The subject treatments using hedgehog antagonists can be effective forboth human and animal subjects. Animal subjects to which the inventionis applicable extend to both domestic animals and livestock, raisedeither as pets or for commercial purposes. Examples are dogs, cats,cattle, horses, sheep, hogs and goats.

II. Definitions

For convience, certain terms employed in the specfication, examples, andappended claims are collected here.

The term “hedgehog polypeptide” encompasses preparations of hedgehogproteins and peptidyl fragments thereof, both agonist and antagonistforms as the specific context will make clear.

An “effective amount” of, e.g., a hedgehog antagonist, with respect tothe subject method of treatment, refers to an amount of the antagonistin a preparation which, when applied as part of a desired dosage regimenbrings about, e.g., a change in the rate of cell proliferation and/orthe state of differentiation of a cell and/or rate of survival of a cellaccording to clinically acceptable standards for the disorder to betreated or the cosmetic purpose.

A “patient” or “subject” to be treated by the subject method can meaneither a human or non-human animal.

The “growth state” of a cell refers to the rate of proliferation of thecell and/or the state of differentiation of the cell.

The terms “steroid” and “'steroid-like” are used interchangeable hereinand refer to a general class of polycyclic compounds possessing theskeleton of cyclopentanophenanthrene or a skeleton derived therefrom byone or more bond scissions or ring expansions or contractions. The ringsmay be substituted at one or more positions, to create derivatives thatadhere to the rules of valence and stability, such as by methyl or otherlower alkyl groups, hydroxyl groups, alkoxyl groups and the like.

The terms “epithelia”, “epithelial” and “epithelium” refer to thecellular covering of internal and external body surfaces (cutaneous,mucous and serous), including the glands and other structures derivedtherefrom, e.g., corneal, esophegeal, epidermal, and hair follicleepithelial cells. Other exemplary epithlelial tissue includes: olfactoryepithelium, which is the pseudostratified epithelium lining theolfactory region of the nasal cavity, and containing the receptors forthe sense of smell; glandular epithelium, which refers to epitheliumcomposed of secreting cells; squamous epithelium, which refers toepithelium composed of flattened plate-like cells. The term epitheliumcan also refer to transitional epithelium, like that which ischaracteristically found lining hollow organs that are subject to greatmechanical change due to contraction and distention, e.g. tissue whichrepresents a transition between stratified squamous and columnarepithelium.

The term “epithelialization” refers to healing by the growth ofepithelial tissue over a denuded surface.

The term “skin” refers to the outer protective covering of the body,consisting of the corium and the epidermis and is understood to includesweat and sebaceous glands, as well as hair follicle structures.Throughout the present application, the adjective “cutaneous” may beused, and should be understood to refer generally to attributes of theskin, as appropriate to the context in which they are used.

The term “epidermis” refers to the outermost and nonvascular layer ofthe skin, derived from the embryonic ectoderm, varying in thickness from0.07-1.4 mm. On the palmar and plantar surfaces it comprises, fromwithin outward, five layers; basal layer composed of columnar cellsarranged perpendicularly; prickle-cell or spinous layer composed offlattened polyhedral cells with short processes or spines; granularlayer composed of flattened granular cells; clear layer composed ofseveral layers of clear, transparent cells in which the nuclei areindistinct or absent; and horny layer composed of flattened, cornifiednon-nucleated cells. In the epidermis of the general body surface, theclear layer is usually absent.

The “corium” or “dermis” refers to the layer of the skin deep to theepidermis, consisting of a dense bed of vascular connective tissue, andcontaining the nerves and terminal organs of sensation. The hair roots,and sebaceous and sweat glands are structures of the epidermis which aredeeply embedded in the dermis.

The term “nail” refers to the horny cutaneous plate on the dorsalsurface of the distal end of a finger or toe.

The term “epidermal gland” refers to an aggregation of cells associatedwith the epidermis and specialized to secrete or excrete materials notrelated to their ordinary metabolic needs. For example, “sebaceousglands” are holocrine glands in the corium that secrete an oilysubstance and sebum. The term “sweat glands” refers to glands thatsecrete sweat, situated in the corium or subcutaneous tissue, opening bya duct on the body surface.

The term “hair” refers to a threadlike structure, especially thespecialized epidermal structure composed of keratin and developing froma papilla sunk in the corium, produced only by mammals andcharacteristic of that group of animals. Also, the aggregate of suchhairs. A “hair follicle” refers to one of the tubular-invaginations ofthe epidermis enclosing the hairs, and from which the hairs grow; and“hair follicle epithelial cells” refers to epithelial cells whichsurround the dermal papilla in the hair follicle, e.g. stem cells, outerroot sheath cells, matrix cells, and inner root sheath cells. Such cellsmay be normal non-malignant cells, or transformed/immortalized cells.

The term “nasal epithelial tissue” refers to nasal and olfactoryepithelium.

“Excisional wounds” include tears, abrasions, cuts, punctures orlacerations in the epithelial layer of the skin and may extend into thedermal layer and even into subcutaneous fat and beyond. Excisionalwounds can result from surgical procedures or from accidentalpenetration of the skin.

“Burn wounds” refer to cases where large surface areas of skin have beenremoved or lost from an individual due to heat and/or chemical agents.

“Dermal skin ulcers” refer to lesions on the skin caused by superficialloss of tissue, usually with inflammation. Dermal skin ulcers which canbe treated by the method of the present invention include decubitusulcers, diabetic ulcers, venous stasis ulcers and arterial ulcers.Decubitus wounds refer to chronic ulcers that result from pressureapplied to areas of the skin for extended periods of time. Wounds ofthis type are often called bedsores or pressure sores. Venous stasisulcers result from the stagnation of blood or other fluids fromdefective veins. Arterial ulcers refer to necrotic skin in the areaaround arteries having poor blood flow.

“Dental tissue” refers to tissue in the mouth which is similar toepithelial tissue, for example gum tissue. The method of the presentinvention is useful for treating periodontal disease.

“Internal epithelial tissue” refers to tissue inside the body which hascharacteristics similar to the epidermal layer in the skin. Examplesinclude the lining of the intestine. The method of the present inventionis useful for promoting the healing of certain internal wounds, forexample wounds resulting from surgery.

A “wound to eye tissue” refers to severe dry eye syndrome, cornealulcers and abrasions and ophthalmic surgical wounds.

Throughout this application, the term “proliferative skin disorder”refers to any disease/disorder of the skin marked by unwanted oraberrant proliferation of cutaneous tissue. These conditions aretypically characterized by epidermal cell proliferation or incompletecell differentiation, and include, for example, X-linked ichthyosis,psoriasis, atopic dermatitis, allergic contact dermatitis, epidermolytichyperkeratosis, and seborrheic dermatitis. For example,epidermodysplasia is a form of faulty development of the epidermis.Another example is “epidermolysis”, which refers to a loosened state ofthe epidermis with formation of blebs and bullae either spontaneously orat the site of trauma.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate surrounding tissues and to giverise to metastases. Exemplary carcinomas include: “basal cellcarcinoma”, which is an epithelial tumor of the skin that, while seldommetastasizing, has potentialities for local invasion and destruction;“squamous cell carcinoma”, which refers to carcinomas arising fromsquamous epithelium and having cuboid cells; “carcinosarcoma”, whichinclude malignant tumors composed of carcinomatous and sarcomatoustissues; “adenocystic carcinoma”, carcinoma marked by cylinders or bandsof hyaline or mucinous stroma separated or surrounded by nests or cordsof small epithelial cells, occurring in the mammary and salivary glands,and mucous glands of the respiratory tract; “epidermoid carcinoma”,which refers to cancerous cells which tend to differentiate in the sameway as those of the epidermis; i.e., they tend to form prickle cells andundergo cornification; “nasopharyngeal carcinoma”, which refers to amalignant tumor arising in the epithelial lining of the space behind thenose; and “renal cell carcinoma”, which pertains to carcinoma of therenal parenchyma composed of tubular cells in varying arrangements.Another carcinomatous epithelial growth is “papillomas”, which refers tobenign tumors derived from epithelium and having a papillomavirus as acausative agent; and “epidermoidomas”, which refers to a cerebral ormeningeal tumor formed by inclusion of ectodermal elements at the timeof closure of the neural groove.

As used herein, the term “psoriasis” refers to a hyperproliferative skindisorder which alters the skin's regulatory mechanisms. In particular,lesions are formed which involve primary and secondary alterations inepidermal proliferation, inflammatory responses of the skin, and anexpression of regulatory molecules such as lymphokines and inflammatoryfactors. Psoriatic skin is morphologically characterized by an increasedturnover of epidermal cells, thickened epidermis, abnormalkeratinization, inflammatory cell infiltrates into the dermis layer andpolymorphonuclear leukocyte infiltration into the epidermis layerresulting in an increase in the basal cell cycle. Additionally,hyperkeratotic and parakeratotic cells are present.

The term “keratosis” refers to proliferative skin disorder characterizedby hyperplasia of the horny layer of the epidermis. Exemplary keratoticdisorders include keratosis follicularis, keratosis palmaris etplantaris, keratosis pharyngea, keratosis pilaris, and actinickeratosis.

As used herein, “proliferating” and “proliferation” refer to cellsundergoing mitosis.

As used herein, “transformed cells” refers to cells which havespontaneously converted to a state of unrestrained growth, i.e., theyhave acquired the ability to grow through an indefinite number ofdivisions in culture. Transformed cells may be characterized by suchterms as neoplastic, anaplastic and/or hyperplastic, with respect totheir loss of growth control.

As used herein, “immortalized cells” refers to cells which have beenaltered via chemical and/or recombinant means such that the cells havethe ability to grow through an indefinite number of divisions inculture.

The term “prodrug” is intended to encompass compounds which, underphysiological conditions, are converted into the therapeutically activeagents of the present invention. A common method for making a prodrug isto select moieties which are hydrolyzed under physiological conditionsto provide the desired. In other embodiments, the prodrug is convertedby an enzymatic activity of the host animal.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are boron, nitrogen,oxygen, phosphorus, sulfur and selenium.

Herein, the term “aliphatic group” refers to a straight-chain,branched-chain, or cyclic aliphatic hydrocarbon group and includessaturated and unsaturated aliphatic groups, such as an alkyl group, analkenyl group, and an alkynyl group.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ for branchedchain), and more preferably 20 or fewer. Likewise, preferred cycloalkylshave from 3-10 carbon atoms in their ring structure, and more preferablyhave 5, 6 or 7 carbons in the ring structure.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents caninclude, for example, a halogen, a hydroxyl, a carbonyl (such as acarboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (suchas a thioester, a thioacetate, or a thioformate), an alkoxyl, aphosphoryl, a phosphonate, a phosphinate, an amino, an amido, anamidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, analkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromaticmoiety. It will be understood by those skilled in the art that themoieties substituted on the hydrocarbon chain can themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CF₃, —CN andthe like. Exemplary substituted alkyls are described below. Cycloalkylscan be further substituted with alkyls, alkenyls, alkoxys, alkylthios,aminoalkyls, carbonyl-substituted alkyls, —CF₃, —CN, and the like.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g. an aromatic or heteroaromatic group).

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Throughout the application, preferred alkylgroups are lower alkyls. In preferred embodiments, a substituentdesignated herein as alkyl is a lower alkyl.

The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics.” The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl,phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazolc, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridinc, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromaticmoiety —CF₃, —CN, or the like.

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀ and R′₁₀ each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R₈, or R₉ and R₁₀ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R₈ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In preferred embodiments, only one of R₉ or R₁₀can be a carbonyl, e.g., R₉, R₁₀ and the nitrogen together do not forman imide. In even more preferred embodiments, R₉ and R₁₀ (and optionallyR′₁₀) each independently represent a hydrogen, an alkyl, an alkenyl, or—(CH₂)_(m)—R₈. Thus, the term “alkylamine” as used herein means an aminegroup, as defined above, having a substituted or unsubstituted alkylattached thereto, i.e., at least one of R₉ and R₁₀ is an alkyl group.

The term “acylamino” is art-recognized and refers to a moiety that canbe represented by the general formula:

wherein R₉ is as defined above, and R′₁₁ represents a hydrogen, analkyl, an alkenyl or —(CH₁₂)_(m)—R₈, where m and R₈ are as definedabove.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉, R₁₀ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R₈, wherein m and R₈ are defined above.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

The term “carbonyl” is art recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₈ or apharmaceutically acceptable salt, R′₁₁ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above. WhereX is an oxygen and R₁₁ or R′₁₁ is not hydrogen, the formula representsan “ester”. Where X is an oxygen, and R₁₁ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR₁₁ is a hydrogen, the formula represents a “carboxylic acid”. Where Xis an oxygen, and R′₁₁ is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiolcarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thiolester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiolcarboxylic acid.” Where X is a sulfur and R₁₁′ ishydrogen, the formula represents a “thiolformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH₂)_(m)—R₈,where m and R₈ are described above.

The term “sulfonate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p- toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₄₁ is as defined above.

The term “sulfonamido” is art recognized and includes a moiety that canbe represented by the general formula:

in which R₉ and R′₁₁ are as defined above.

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₉ and R₁₀ are as defined above.

The terms “sulfoxido” or “sulfinyl”, as used herein, refers to a moietythat can be represented by the general formula:

in which R₄₄ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

A “phosphoryl” can in general be represented by the formula:

wherein Q1 represented S or O, and R₄₆ represents hydrogen, a loweralkyl or an aryl. When used to substitute, e.g. an alkyl, the phosphorylgroup of the phosphorylalkyl can be represented by the general formula:

wherein Q₁ represented S or O, and each R₄₆ independently representshydrogen, a lower alkyl or an aryl, Q₂ represents O, S or N. When Q₁ isan S, the phosphoryl moiety is a “phosphorothioate”.

A “phosphoramidite” can be represented in the general formula:

wherein R₉ and R₁₀ are as defined above, and Q₂ represents O, S or N.

A “phosphonamidite” can be represented in the general formula:

wherein R₉ and R₁₀ are as defined above, Q₂ represents O, S or N, andR₄₈ represents a lower alkyl or an aryl, Q₂ represents O, S or N.

A “selenoalkyl” refers to an alkyl group having a substituted selenogroup attached thereto. Exemplary “selenoethers” which may besubstituted on the alkyl are selected from one of —Se-alkyl,—Se-alkenyl, —Se-alkynyl, and —Se—(CH₂)_(m)—R₈, m and R₈ being definedabove.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

As used herein, the definition of each expression, e.g. alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof (e.g. the ability to inhibit hedgehogsignaling), wherein one or more simple variations of substituents aremade which do not adversely affect the efficacy of the compound. Ingeneral, the compounds of the present invention may be prepared by themethods illustrated in the general reaction schemes as, for example,described below, or by modifications thereof, using readily availablestarting materials, reagents and conventional synthesis procedures. Inthese reactions, it is also possible to make use of variants which arein themselves known, but are not mentioned here.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Alsofor purposes of this invention, the term “hydrocarbon” is contemplatedto include all permissible compounds having at least one hydrogen andone carbon atom. In a broad aspect, the permissible hydrocarbons includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic organic compounds which can besubstituted or unsubstituted.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991).

A list of many of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

The term “ED₅₀” means the dose of a drug which produces 50% of itsmaximum response or effect. Alternatively, the dose which produces apre-determined response in 50% of test subjects or preparations.

The term “LD₅₀” means the dose of a drug which is lethal in 50% of testsubjects.

The term “therapeutic index” refers to the therapeutic index of a drugdefined as LD₅₀/ED₅₀.

The term “agonist”, with respect to hedgehog, refers to a compound thatmimics the action of a native hedgehog protein.

The term “antagonist”, with respect to hedgehog bioactivity, refers to acompound that inhibits hedgehog-mediated signal transduction. In thecontext of the present invention, such antagonists can include compoundswhich mimic the activity of jervine, having such characteristics as theability to disrupt cholesterol homoeostasis such as through inhibitionof sterol trafficking (e.g., a a class 2 inhibitor), the ability to bindto a hedgehog receptor site and inhibit the simultaneous binding ofhedgehog to the receptor, or, by non-competitive and/or allostericeffects of the like, inhibit the response of the cell to hedgehog whichdoes bind.

The term “competitive antagonist” refers to a compound that binds to areceptor site; its effects can be overcome by increased concentration ofthe agonist.

As used herein, “steroid hormone receptor superfamily” refers to theclass of related receptors comprised of glucocorticoid,mineralocorticoid, progesterone, estrogen, estrogen-related, vitamin D3,thyroid, v-erb-A, retinoic acid and E75 (Drosophila) receptors. As usedherein “steroid hormone receptor” refers to members within the steroidhormone receptor superfamily. In higher organisms, the nuclear hormonereceptor superfamily includes approximately a dozen distinct genes thatencode zinc finger transcription factors, each of which is specificallyactivated by binding a ligand such as a steroid, thyroid hormone (T3) orretinoic acid (RA).

III. Exemplary Compounds of the Invention.

As described in further detail below, it is contemplated that thesubject methods can be carried out using a variety of differentsteroidal alkaloids, as well as non-steroidal small molecules, which canbe readily identified, e.g. by such drug screening assays as describedherein. The above notwithstanding, in a preferred embodiment, themethods and compositions of the present invention make use of compoundshaving a steroidal alkaloid ring system. Steroidal alkaloids have afairly complex nitrogen containing nucleus. Two exemplary classes ofsteroidal alkaloids for use in the subject methods are the Solanum typeand the Veratrum type.

There are more than 50 naturally occuring veratrum alkaloids includingveratramine, cyclopamine, cycloposine, jervine, and muldamine occurringin plants of the Veratrum spp. The Zigadenus spp., death camas, alsoproduces several veratrum-type of steroidal alkaloids includingzygacine. In general, many of the veratrum alkaloids (e.g., jervine,cyclopamine and cycloposine) consist of a modified steroid skeletonattached spiro to a furanopiperidine. A typical veratrum-type alkaloidmay be represented by:

An example of the Solanum type is solanidine. This steroidal alkaloid isthe nucleus (i.e. aglycone) for two important glycoalkaloids, solanineand chaconine, found in potatoes. Other plants in the Solanum familyincluding various nightshades, Jerusalem cherries, and tomatoes alsocontain solanum-type glycoalkaloids. Glycoalkaloids are glycosides ofalkaloids. A typical solanum-type alkaloid may be represented by:

Based on these structures, and the possibility that certain unwantedside effects can be reduced by some manipulation of the structure, awide range of steroidal alkaloids are contemplated as potential hedgehogantagonists for use in the subject method. For example, compounds usefulin the subject methods include steroidal alkaloids represented in thegeneral forumlas (I) or unsaturated forms thereof and/or seco-, nor- orhomo-derivatives thereof:

wherein, as valence and stability permit,

R₂, R₃, R₄, and R₅, represent one or more substitutions to the ring towhich each is attached, for each occurrence, independently representhydrogen, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, ═O, ═S,alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides,phosphoryls, phosphonates, phosphines, carbonyls, carboxyls,carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls,arylsulfonyls, selenoethers, ketones, aldehydes, esters, or—(CH₂)_(m)—R₈;

R₆, R₇, and R′₇, are absent or represent, independently, halogens,alkyls, alkenyls, alkynyls, aryls, hydroxyl, ═O, ═S, alkoxyl, silyloxy,amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates,phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls,ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers,ketones, aldehydes, esters, or —(CH₂)_(m)—R₈, or

R₆ and R₇, or R₇ and R′₇, taken together form a ring or polycyclic ring,e.g., which is susbstituted or unsubstituted, with the proviso that atleast one of R₆, R₇, or R′₇ is present and includes a primary orsecondary amine;

R₈ represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle, or apolycycle; and

m is an integer in the range 0 to 8 inclusive.

In preferred embodiments,

R₂ and R₃, for each occurrence, is an —OH, alkyl, —O-alkyl, —C(O)-alkyl,or —C(O)—R₈;

R₄, for each occurrence, is an absent, or represents —OH, ═O, alkyl,—O-alkyl, —C(O)-alkyl, or —C(O)—R₈;

R₆, R₇, and R′₇ each independently represent, hydrogen, alkyls,alkenyls, alkynyls, amines, imines, amides, carbonyls, carboxyls,carboxamides, ethers, thioethers, esters, or —(CH₂)_(m)—R₈, or

R₇, and R′₇ taken together form a furanopiperidine, such asperhydrofuro[3,2-b]pyridine, a pyranopiperidine, a quinoline, an indole,a pyranopyrrole, a naphthyridine, a thiofuranopiperidine, or athiopyranopiperidine

with the proviso that at least one of R₆, R₇, or R′₇ is present andincludes a primary or secondary amine;

R₈ represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle, or apolycycle, and preferably R₈ is a piperidine, pyrimidine, morpholine,thiomorpholine, pyridazine.

In certain preferred embodiments, the definitions outlined above apply,and the subject compounds are represented by general formula Ia orunsaturated forms thereof and/or seco-, nor- or homo-derivativesthereof:

In preferred embodiments, the subject hedgehog antagonists can berepresented in one of the following general formulas (II) or unsaturatedforms thereof and/or seco-, nor- or homo-derivatives thereof:

wherein R₂, R₃, R₄, R₅, R₆, R₇, and R′₇ are as defined above, and Xrepresents O or S, though preferably O.

In certain preferrred embodiments, the definitions outlined above apply,and the subject compounds are represented by general formula IIa orunsaturated forms thereof and/or seco-, nor- or homo-derivativesthereof:

In certain embodiments, the subject hedgehog antagonists are representedby the general formula (III) or unsaturated forms thereof and/or seco-,nor- or homo-derivatives thereof:

wherein

R₂, R₃, R₄, R₅ and R₈ are as defined above;

A and B represent monocyclic or polycyclic groups;

T represent an alkyl, an aminoalkyl, a carboxyl, an ester, an amide,ether or amine linkage of 1-10 bond lengths;

T′ is absent, or represents an alkyl, an aminoalkyl, a carboxyl, anester, an amide, ether or amine linkage of 1-3 bond lengths, wherein ifT and T′ are present together, than T and T′ taken together with thering A or B form a covelently closed ring of 5-8 ring atoms;

R9 represent one or more substitutions to the ring A or B, which foreach occurrence, independently represent halogens, alkyls, alkenyls,alkynyls, aryls, hydroxyl, ═O, ═S, alkoxyl, silyloxy, amino, nitro,thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines,carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers,thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones,aldehydes, esters, or —(CH₂)_(m)—R₉; and

n and m are, independently, zero, 1 or 2;

with the proviso that A and R₉, or T, T′ B and R₉, taken togetherinclude at least one primary or secondary amine.

In certain preferred embodiments, the definitions outlined above apply,and the subject compounds are represented by general formula IIIa orunsaturated forms thereof and/or seco-, nor- or homo-derivativesthereof:

For example, the subject methods can utilize hedgehog antagonists basedon the veratrum-type steroidal alkaloids jervine, cyclopamine,cycloposine, mukiamine or veratramine, e.g., which may be represented inthe general formula (IV) or unsaturated forms thereof and/or seco-, nor-or homo-derivatives thereof:

wherein

R₂, R₃, R₄, R₅, R₆ and R₉ are as defined above;

R₂₂ is absent or represents an alkyl, an alkoxyl or —OH.

In certain preferred embodiments, the definitions outlined above apply,and the subject compounds are represented by general formula IVa orunsaturated forms thereof and/or seco-, nor- or homo-derivativesthereof:

In even more preferred embodments, the subject antagonists arerepresented in the formulas (V) or unsaturated forms thereof and/orseco-, nor- or homo-derivatives thereof:

wherein R₂, R₃, R₄, R₆ and R₉ are as defined above;

In certain preferred embodiments, the definitions outlined above apply,and the subject compounds are represented by general formula Va orunsaturated forms thereof and/or seco-, nor- or homo-derivativesthereof:

Another class of hedgehog antagonists can be based on the veratrum-typesteroidal alkaloids resmebling verticine and zygacine, e.g., representedin the general formulas (VI) or unsaturated forms thereof and/or seco-,nor- or homo-derivatives thereof:

wherein R₂, R₃, R₄, R₅ and R₉ are as defined above.

In certain preferred embodiments, the definitions outlined above apply,and the subject compounds are represented by general formula VIa orunsaturated forms thereof and/or seco-, nor- or homo-derivativesthereof:

Still another class of potential hedgehog antagonists are based on thesolanum-type steroidal alkaloids, e.g., solanidine, which may berepresented in the general formula (VII) or unsaturated forms thereofand/or seco-, nor- or homo-derivatives thereof:

wherein R₂, R₃, R₄, R₅ and R₉ are as defined above.

In certain preferred embodiments, the definitions outlined above apply,and the subject compounds are represented by general formula VIIa orunsaturated forms thereof and/or seco-, nor- or homo-derivativesthereof:

In certain embodiments, the subject antagonists can be chosen on thebasis of their selectively for the hedgehog pathway. This selectivitycan for the hedgehog pathway versus other steroid-mediated pathways(such as testosterone or estrogen mediated activities), as well asselectivity for particular hedgehog pathways, e.g., which isotypespecific for hedgehog (e.g., Shh. Ihh, Dhh) or the patched receptor(e.g., ptc-1, ptc-2). For instance, the subject method may employsteroidal alkaloids which do not substantially interfere with thebiological activity of such steroids as aldosterone, androstane,androstene, androstenedione, androsterone, cholecalciferol, cholestane,cholic acid corticosterone, cortisol, cortisol acetate, cortisone,cortisone acetate, deoxycorticosterone, digitoxigenin, ergocalciferol,ergosterol, estradiol-17-α, estradiol-17-β, estriol, estrane, estrone,hydrocortisone, lanosterol, lithocholic acid, mestranol, β-methasone,prednisone, pregnane, pregnenolone, progesterone, spironolactone,testosterone, triamcinolone and their derivatives, at least so far asthose activities are unrelated to ptc related signaling.

In one embodiment, the subject steroidal alkaloid for use in the presentmethod has a k_(d) for members of the nuclear hormone receptorsuperfamily of greater than 1 μM, and more preferably greater than 1 mM,e.g., it does not bind estrogen., testosterone receptors or the like.Preferably, the subject hedgehog antagonist has no estrogenic activityat physiological concentrations (e.g., in the range of 1 ng-1 mg/kg).

In this manner, untoward side effects which may be associated certainmembers of the steroidal alkaloid class can be reduced. For example,using the drug screening assays described herein, the application ofcombinatorial and medicinal chemistry techniques to the steroidalalkaloids provides a means for reducing such unwanted negative sideeffects including personality changes, shortened life spans,cardiovascular diseases and vascular occlusion., organ toxicity,hyperglycemia and diabetes, Cushnoid features, “wasting” syndrome,steroidal glaucoma, hypertension, peptic ulcers, and increasedsusceptibility to infections. For certain embodiments, it will bebenefical to reduce the teratogenic activity relative to jervine, as forexample, in the use of the subject method to selectively inhibitspermatogenesis.

In preferred embodiment, the subject antagonists are steroidal alkaloidsother than spirosolane, tomatidine, jervine, etc.

In certain preferred embodiments, the subject inhibitors inhibithedgehog-mediated signal transduction with an ED₅₀ of 1 mM or less, morepreferably of 1 μM or less, and even more preferably of 1 nM or less.

In certain embodiments, the subject inhibitors inhibit hedgehog-mediatedsignal transduction with an ED₅₀ of 1 mM or less, more preferably 1 μMor less, and even more preferably 1 nM or less.

In particular embodiments, the steroidal alkaloid is chosen for usebecause it is more selective for one hedgehog isoform over the next,e.g., 10 fold, and more preferably at least 100 or even 1000 fold moreselective for one hedgehog pathway (Shh, Ihh, Dhh) over another.Likewise, a steroidal alkaloid can be chosen for use because it is moreselective for one patched isoform over the next, e.g., 10 fold, and morepreferably at least 100 or even 1000 fold more selective for one patchedpathway (ptc-1, ptc-2) over another.

IV. Exemplary Applications of Method and Compositions

Another aspect of the present invention relates to a method ofmodulating a differentiated state, survival, and/or proliferation of acell responsive to a hedgehog protein, by contacting the cells with ahedgehog antagonist according to the subject method and as thecircumstances may warrant. For instance, it is contemplated by theinvention that, in light of the present finding of an apparently broadinvolvement of hedgehog proteins in the formation of ordered spatialarrangements of differentiated tissues in vertebrates, the subjectmethod could be used as part of a process for generating and/ormaintaining an array of different vertebrate tissue both in vitro and invivo. The hedgehog antagonist, whether inductive or anti-inductive withrespect proliferation or differentiation of a given tissue, can be, asappropriate, any of the preparations described above, includingveratrum-type alkaloids and solanum-type alkaloids.

For example, the present method is applicable to cell culturetechniques. In vitro neuronal culture systems have proved to befundamental and indispensable tools for the study of neural development,as well as the identification of neurotrophic factors such as nervegrowth factor (NGF), ciliary trophic factors (CNTF), and brain derivedneurotrophic factor (BDNF). Once a neuronal cell has becometerminally-differentiated it typically will not change to anotherterminally differentiated cell-type. However, neuronal cells cannevertheless readily lose their differentiated state. This is commonlyobserved when they are grown in culture from adult tissue, and when theyform a blastema during regeneration. The present method provides a meansfor ensuring an adequately restrictive environment in order to maintainneuronal cells at various stages of differentiation, and can beemployed, for instance, in cell cultures designed to test the specificactivities of other trophic factors. In such embodiments of the subjectmethod, the cultured cells can be contacted with a hedgehog antagonistof the present invention in order to alter the rate of proliferation ofneuronal stem cells in the culture and/or alter the rate ofdifferentiation, or to maintain the integrity of a culture of certainterminally-differentiated neuronal cells by preventing loss ofdifferentiation. In an exemplary embodiment, the subject method can beused to culture, for example, sensory neurons or, alternatively,motorneurons. Such neuronal cultures can be used as convenient assaysystems as well as sources of implantable cells for therapeutictreatments. For example, hedgehog polypeptides may be useful inestablishing and maintaining the olfactory neuron cultures described inU.S. Pat. No. 5,318,907 and the like.

According to the present invention, large numbers of non-tumorigenicneural progenitor cells can be perpetuated in vitro and their rate ofproliferation and/or differentiation can be effected by contact withhedgehog antagonists of the present invention. Generally, a method isprovided comprising the steps of isolating neural progenitor cells froman animal, perpetuating these cells in vitro or in vivo, preferably inthe presence of growth factors, and regulating the differentiation ofthese cells into particular neural phenotypes, e.g., neurons and glia,by contacting the cells with a hedgehog antagonist.

Progenitor cells are thought to be under a tonic inhibitory influencewhich maintains the progenitors in a suppressed state until theirdifferentiation is required. However, recent techniques have beenprovided which permit these cells to be proliferated, and unlike neuronswhich are terminally differentiated and therefore non-dividing, they canbe produced in unlimited number and are highly suitable fortransplantation into heterologous and autologous hosts withneurodegenerative diseases.

By “progenitor” it is meant an oligopotent or multipotent stem cellwhich is able to divide without limit and, under specific conditions,can produce daughter cells which terminally differentiate such as intoneurons and glia. These cells can be used for transplantation into aheterologous or autologous host. By heterologous is meant a host otherthan the animal from which the progenitor cells were originally derived.By autologous is meant the identical host from which the cells wereoriginally derived.

Cells can be obtained from embryonic, post-natal, juvenile or adultneural tissue from any animal. By any animal is meant any multicellularanimal which contains nervous tissue. More particularly, is meant anyfish, reptile, bird, amphibian or mammal and the like. The mostpreferable donors are mammals, especially mice and humans.

In the case of a heterologous donor animal, the animal may beeuthanized, and the brain and specific area of interest removed using asterile procedure. Brain areas of particular interest include any areafrom which progenitor cells can be obtained which will serve to restorefunction to a degenerated area of the host's brain. These regionsinclude areas of the central nervous system (CNS) including the cerebralcortex, cerebellum, midbrain, brainstem, spinal cord and ventriculartissue, and areas of the peripheral nervous system (PNS) including thecarotid body and the adrenal medulla. More particularly, these areasinclude regions in the basal ganglia, preferably the striatum whichconsists of the caudate and putamen, or various cell groups such as theglobus pallidus, the subthalamic nucleus, the nucleus basalis which isfound to be degenerated in Alzheimer's Disease patients, or thesubstantia nigra pars compacta which is found to be degenerated inParkinson's Disease patients.

Human heterologous neural progenitor cells may be derived from fetaltissue obtained from elective abortion, or from a post-natal, juvenileor adult organ donor. Autologous neural tissue can be obtained bybiopsy, or from patients undergoing neurosurgery in which neural tissueis removed, in particular during epilepsy surgery, and more particularlyduring temporal lobectomies and hippocampalectomies.

Cells can be obtained from donor tissue by dissociation of individualcells from the connecting extracellular matrix of the tissue.Dissociation can be obtained using any known procedure, includingtreatment with enzymes such as trypsin, collagenase and the like, or byusing physical methods of dissociation such as with a blunt instrument.Dissociation of fetal cells can be carried out in tissue culture medium,while a preferable medium for dissociation of juvenile and adult cellsis artificial cerebral spinal fluid (aCSF). Regular aCSF contains 124 mMNaCl, 5 mM KCl, 1.3 mM MgCl₂, 2 mM CaCl₂, 26 mM NaHCO₃, and 10 mMD-glucose. Low Ca²⁺ aCSF contains the same ingredients except for MgCl₂at a concentration of 3.2 mM and CaCl₂ at a concentration of 0.1 mm.

Dissociated cells can be placed into any known culture medium capable ofsupporting cell growth, including MEM, DMEM, RPMI, F-12. and the like,containing supplements which are required for cellular metabolism suchas glutamine and other amino acids, vitamins, minerals and usefulproteins such as transferrin and the like. Medium may also containantibiotics to prevent contamination with yeast, bacteria and fungi suchas penicillin, streptomycin, gentamicin and the like. In some cases, themedium may contain serum derived from bovine, equine, chicken and thelike. A particularly preferable medium for cells is a mixture of DMEMand F-12.

Conditions for culturing should be close to physiological conditions.The pH of the culture media should be close to physiological pH,preferably between pH 6-8, more preferably close to pH 7. even moreparticularly about pH 7.4. Cells should be cultured at a temperatureclose to physiological temperature, preferably between 30° C.-40° C.,more preferably between 32° C.-38° C., and most preferably between 35°C.-37° C.

Cells can be grown in suspension or on a fixed substrate, butproliferation of the progenitors is preferably done in suspension togenerate large numbers of cells by formation of “neurospheres” (see, forexample, Reynolds et al. (1992) Science 255:1070-1709; and PCTPublications WO93/01275, WO94/09119, WO94/10292, and WO94/16718). In thecase of propagating (or splitting) suspension cells, flasks are shakenwell and the neurospheres allowed to settle on the bottom corner of theflask. The spheres are then transferred to a 50 ml centrifuge tube andcentrifuged at low speed. The medium is aspirated, the cells resuspendedin a small amount of medium with growth factor, and the cellsmechanically dissociated and resuspended in separate aliquots of media.

Cell suspensions in culture medium are supplemented with any growthfactor which allows for the proliferation of progenitor cells and seededin any receptacle capable of sustaining cells, though as set out above,preferably in culture flasks or roller bottles. Cells typicallyproliferate within 3-4 days in a 37° C. incubator, and proliferation canbe reinitiated at any time after that by dissociation of the cells andresuspension in fresh medium containing growth factors.

In the absence of substrate, cells lift off the floor of the flask andcontinue to proliferate in suspension forming a hollow sphere ofundifferentiated cells. After approximately 3-10 days in vitro, theproliferating clusters (neurospheres) are fed every 2-7 days, and moreparticularly every 2-4 days by gentle centrifugation and resuspension inmedium containing growth factor.

After 6-7 days in vitro, individual cells in the neurospheres can beseparated by physical dissociation of the neurospheres with a bluntinstrument, more particularly by triturating the neurospheres with apipette. Single cells from the dissociated neurospheres are suspended inculture medium containing growth factors, and differentiation of thecells can be control in culture by plating (or resuspending) the cellsin the presence of a hedgehog antagonist.

To further illustrate other uses of the subject hedgehog antagonists, itis noted that intracerebral grafting has emerged as an additionalapproach to central nervous system therapies. For example, one approachto repairing damaged brain tissues involves the transplantation of cellsfrom fetal or neonatal animals into the adult brain (Dunnett et al.(1987) J Exp Biol 123:265-289; and Freund et al. (1985) J Neurosci5:603-616). Fetal neurons from a variety of brain regions can besuccessfully incorporated into the adult brain, and such grafts canalleviate behavioral defects. For example, movement disorder induced bylesions of dopaminergic projections to the basal ganglia can beprevented by grafts of embryonic dopaminergic neurons. Complex cognitivefunctions that are impaired after lesions of the neocortex can also bepartially restored by grafts of embryonic cortical cells. The subjectmethod can be used to regulate the growth state in the culture, or wherefetal tissue is used, especially neuronal stem cells, can be used toregulate the rate of differentiation of the stem cells.

Stem cells useful in the present invention are generally known. Forexample, several neural crest cells have been identified, some of whichare multipotent and likely represent uncommitted neural crest cells, andothers of which can generate only one type of cell, such as sensoryneurons, and likely represent committed progenitor cells. The role ofhedgehog antagonsits employed in the present method to culture such stemcells can be to regulate differentiation of the uncommitted progenitor,or to regulate further restriction of the developmental fate of acommitted progenitor cell towards becoming a terminally-differentiatedneuronal cell. For example, the present method can be used in vitro toregulate the differentiation of neural crest cells into glial cells,schwann cells, chromaffin cells, cholinergic sympathetic orparasympathetic neurons, as well as peptidergic and serotonergicneurons. The hedgehog antagonists can be used alone, or can be used incombination with other neurotrophic factors which act to moreparticularly enhance a particular differentiation fate of the neuronalprogenitor cell.

In addition to the implantation of cells cultured in the presence of thesubject hedgehog antagonists, yet another aspect of the presentinvention concerns the therapeutic application of a hedgehog antagonistto regulate the growth state of neurons and other neuronal cells in boththe central nervous system and the peripheral nervous system. Theability of hedgehog protein to regulate neuronal differentiation duringdevelopment of the nervous system and also presumably in the adult stateindicates that, in certain instances, the subject hedgehog antagonistscan be expected to facilitate control of adult neurons with regard tomaintenance, functional performance, and aging of normal cells; repairand regeneration processes in chemically or mechanically lesioned cells;and treatment of degeneration in certain pathological conditions. Inlight of this understanding, the present invention specificallycontemplates applications of the subject method to the treatmentprotocol of (prevention and/or reduction of the severity of)neurological conditions deriving from: (i) acute, subacute, or chronicinjury to the nervous system, including traumatic injury, chemicalinjury, vascular injury and deficits (such as the ischemia resultingfrom stroke), together with infectious/inflammatory and tumor-inducedinjury; (ii) aging of the nervous system including Alzheimer's disease;(iii) chronic neurodegenerative diseases of the nervous system,including Parkinson's disease, Huntington's chorea, amylotrophic lateralsclerosis and the like, as well as spinocerebellar degenerations; and(iv) chronic immunological diseases of the nervous system or affectingthe nervous system, including multiple sclerosis. The subjectantagonists can be used in conjunction with a therapy involving hedgehogagonists to control the timing and rates of proliferation and/ordifferentiation of the affected neuronal cells.

As appropriate, the subject method can also be used in generating nerveprostheses for the repair of central and peripheral nerve damage. Inparticular, where a crushed or severed axon is intubulated by use of aprosthetic device, hedgehog agonists and antagonists can be added to theprosthetic device to regulate the rate of growth and regeneration of thedendridic processes. Exemplary nerve guidance channels are described inU.S. Pat. Nos. 5,092,871 and 4,955,892.

In another embodiment, the subject method can be used in the treatmentof neoplastic or hyperplastic transformations such as may occur in thecentral nervous system. For instance, the hedgehog antagonists can beutilized to cause such transformed cells to become either post-mitoticor apoptotic. The present method may, therefore, be used as part of atreatment for, e.g., malignant gliomas, medulloblastomas,neuroectodermal tumors, and ependymomas.

Yet another aspect of the present invention concerns the observation inthe art that hedgehog proteins are morphogenic signals involved in othervertebrate organogenic pathways in addition to neuronal differentiationas described above, having apparent roles in other endodermalpatterning, as well as both mesodermal and endodermal differentiationprocesses. Thus, it is contemplated by the invention that compositionscomprising hedgehog antagonists can also be utilized for both cellculture and therapeutic methods involving generation and maintenance ofnon-neuronal tissue.

In one embodiment, the present invention makes use of the discovery thathedgehog proteins, such as Sonic hedgehog, are apparently involved incontrolling the development of stem cells responsible for formation ofthe digestive tract, liver, lungs, and other organs which derive fromthe primitive gut. Shh serves as an inductive signal from the endodermto the mesoderm, which is critical to gut morphogenesis. Therefore, forexample, hedgehog antagonists of the instant method can be employed forregulating the development and maintenance of an artificial liver whichcan have multiple metabolic functions of a normal liver. In an exemplaryembodiment, the subject method can be used to regulate the proliferationand differentiation of digestive tube stem cells to form hepatocytecultures which can be used to populate extracellular matrices, or whichcan be encapsulated in biocompatible polymers, to form both implantableand extracorporeal artificial livers.

In another embodiment, therapeutic compositions of hedgehog agonists canbe utilized in conjunction with transplantation of such artificiallivers, as well as embryonic liver structures, to regulate uptake ofintraperitoneal implantation, vascularization, and in vivodifferentiation and maintenance of the engrafted liver tissue.

In yet another embodiment, the subject method can be employedtherapeutically to regulate such organs after physical, chemical orpathological insult. For instance, therapeutic compositions comprisinghedgehog antagonists can be utilized in liver repair subsequent to apartial hepatectomy.

The generation of the pancreas and small intestine from the embryonicgut depends on intercellular signalling between the endodermal andmesodermal cells of the gut. In particular, the differentiation ofintestinal mesoderm into smooth muscle has been suggested to depend onsignals from adjacent endodermal cells. One candidate mediator ofendodermally derived signals in the embryonic hindgut is Sonic hedgehog.See, for example, Apclqvist et al. (1997) Curr Biol 7:801-4. The Shhgene is expressed throughout the embryonic gut endoderm with theexception of the pancreatic bud endoderm, which instead expresses highlevels of the homeodomain protein Ipf1/Pdx1 (insulin promoter factor1/pancreatic and duodenal homcobox 1), an essential regulator of earlypancreatic development. Apclqvist et al., supra, have examined whetherthe differential expression of Shh in the embryonic gut tube controlsthe differentiation of the surrounding mesoderm into specialisedmesoderm derivatives of the small intestine and pancreas. To test this,they used the promoter of the Ipf1/Pdx1 gene to selectively express Shhin the developing pancreatic epithelium. In Ipf1/Pdx1-Shh transgenicmice, the pancreatic mesoderm developed into smooth muscle andinterstitial cells of Cajal, characteristic of the intestine, ratherthan into pancreatic mesenchyme and spleen. Also, pancreatic explantsexposed to Shh underwent a similar program of intestinaldifferentiation. These results provide evidence that the differentialexpression of endodermally derived Shh controls the fate of adjacentmesoderm at different regions of the gut tube.

In the context of the present invention, it is contemplated thereforethat the subject hedgehog inhibitors can be used to control the regulatethe proliferation and/or differentiation of pancreatic tissue both invivo and in vitro.

There are a wide variety of pathological cell proliferative anddifferentiative conditions for which the inhibitors of the presentinvention may provide therapeutic benefits, with the general strategybeing, for example, the correction of abberrant insulin expression, ormodulation of differentiation. More generally, however, the presentinvention relates to a method of inducing and/or maintaining adifferentiated state, enhancing survival and/or affecting proliferationof pancreatic cells, by contacting the cells with the subjectinhibitors. For instance, it is contemplated by the invention that, inlight of the apparent involvement of hedgehog protein(s) in theformation of ordered spatial arrangements of pancreatic tissues, thesubject method could be used as part of a technique to generate and/ormaintain such tissue both in vitro and in vivo. For instance, modulationof the function of ptc can be employed in both cell culture andtherapeutic methods involving generation and maintenance β-cells andpossibly also for non-pancreatic tissue, such as in controlling thedevelopment and maintenance of tissue from the digestive tract, spleen,lungs, and other organs which derive from the primitive gut.

In an exemplary embodiment, the present method can be used in thetreatment of hyperplastic and neoplastic disorders effecting pancreatictissue, particularly those characterized by aberrant proliferation ofpancreatic cells. For instance, pancreatic cancers are marked byabnormal proliferation of pancreatic cells which can result inalterations of insulin secretory capacity of the pancreas. For instance,certain pancreatic hyperplasias, such as pancreatic carcinomas, canresult in hypoinsulinemia due to dysfunction of β-cells or decreasedislet cell mass. To the extent that aberrant hedgehog signaling may beindicated in disease progression, the subject inhibitors, can be used toenhance regeneration of the tissue after anti-tumor therapy.

Moreover, manipulation of ptc signaling properties at different pointsmay be useful as part of a strategy for reshaping/repairing pancreatictissue both in vivo and in vitro. In one embodiment, the presentinvention makes use of the apparent involvement of the hedgehog inregulating the development of pancreatic tissue. In general, the subjectmethod can be employed therapeutically to regulate the pancreas afterphysical, chemical or pathological insult. In yet another embodiment,the subject method can be applied to to cell culture techniques, and inparticular, may be employed to enhance the initial generation ofprosthetic pancreatic tissue devices. Manipulation of proliferation anddifferentiation of pancreatic tissue, for example, by altering ptcactivity, can provide a means for more carefully controlling thecharacteristics of a cultured tissue. In an exemplary embodiment, thesubject method can be used to augment production of prosthetic deviceswhich require β-islet cells, such as may be used in the encapsulationdevices described in, for example, the Aebischer et al. U.S. Pat. No.4,892,538, the Aebischer et al. U.S. Pat. No. 5,106,627, the Lim U.S.Pat. No. 4,391,909, and the Sefton U.S. Pat. No. 4,353,888. Earlyprogenitor cells to the pancreatic islets are multipotential, andapparently coactive all the islet-specific genes from the time theyfirst appear. As development proceeds, expression of islet-specifichormones, such as insulin, becomes restricted to the pattern ofexpression characteristic of mature islet cells. The phenotype of matureislet cells, however, is not stable in culture, as reappearence ofembyonal traits in mature β-cells can be observed. By utilizing agentswhich alter ptc signal transduction, the the action of endogenoushedgehog protein on the differentiation path or proliferative index ofthe cells can be regulated.

Furthermore, manipulation of the differentiative state of pancreatictissue can be utilized in conjunction with transplantation of artificialpancreas so as to promote implantation, vascularization, and in vivodifferentiation and maintenance of the engrafted tissue. For instance,manipulation of ptc function to affect tissue differentiation can beutilized as a means of maintaining graft viability.

Bellusci et al. (1997) Development 124:53 report that Sonic hedgehogregulates lung mesenchymal cell proliferation in vivo. Accordingly, thepresent method can be used to regulate regeneration of lung tissue,e.g., in the treatment of emphysema.

Fujita et al. (1997) Biochem Biophys Res Commun 238:658 reported thatSonic hedgehog is expressed in human lung squamous carcinoma andadenocarcinoma cells. The expression of Sonic hedgehog was also detectedin the human lung squamous carcinoma tissues, but not in the normal lungtissue of the same patient. They also observed that Sonic hedgehogstimulates the incorporation of BrdU into the carcinoma cells andstimulates their cell growth, while anti-Shh-N inhibited their cellgrowth. These results suggest that a Sonic hedgehog signal is involvedin the cell growth of such transformed lung tissue and thereforeindicates that the subject method can be used as part of a treatment oflung carcinoma and adenocarcinomas, and other proliferative disordersinvolving the lung epithelia.

In still another embodiment of the present invention, compositionscomprising hedgehog antagonists can be used in the in vitro generationof skeletal tissue, such as from skeletogenic stem cells, as well as thein vivo treatment of skeletal tissue deficiencies. The present inventionparticularly contemplates the use of hedgehog antagonists to regulatethe rate of chondrogenesis and/or osteogenesis. By “skeletal tissuedeficiency”, it is meant a deficiency in bone or other skeletalconnective tissue at any site where it is desired to restore the bone orconnective tissue, no matter how the deficiency originated, e.g. whetheras a result of surgical intervention, removal of tumor, ulceration,implant, fracture, or other traumatic or degenerative conditions.

For instance, the method of the present invention can be used as part ofa regimen for restoring cartilage function to a connective tissue. Suchmethods are useful in, for example, the repair of defects or lesions incartilage tissue which is the result of degenerative wear such as thatwhich results in arthritis, as well as other mechanical derangementswhich may be caused by trauma to the tissue, such as a displacement oftorn meniscus tissue, meniscectomy, a laxation of a joint by a tornligament, malignment of joints, bone fracture, or by hereditary disease.The present reparative method is also useful for remodeling cartilagematrix, such as in plastic or reconstructive surgery, as well asperiodontal surgery. The present method may also be applied to improvinga previous reparative procedure, for example, following surgical repairof a meniscus, ligament, or cartilage. Furthermore, it may prevent theonset or exacerbation of degenerative disease if applied early enoughafter trauma.

In one embodiment of the present invention, the subject method comprisestreating the afflicted connective tissue with a therapeuticallysufficient amount of a hedgehog antagonist, particularly an antagonistselective for Indian hedgehog, to regulate a cartilage repair responsein the connective tissue by managing the rate of differentiation and/orproliferation of chondrocytes embedded in the tissue. Such connectivetissues as articular cartilage, interarticular cartilage (menisci),costal cartilage (connecting the true ribs and the sternum), ligaments,and tendons are particularly amenable to treatment in reconstructiveand/or regenerative therapies using the subject method. As used herein,regenerative therapies include treatment of degenerative states whichhave progressed to the point of which impairment of the tissue isobviously manifest, as well as preventive treatments of tissue wheredegeneration is in its earliest stages or imminent.

In an illustrative embodiment, the subject method can be used as part ofa therapeutic intervention in the treatment of cartilage of adiarthroidal joint, such as a knee, an ankle, an elbow, a hip, a wrist,a knuckle of either a finger or toe, or a tempomandibular joint. Thetreatment can be directed to the meniscus of the joint, to the articularcartilage of the joint, or both. To further illustrate, the subjectmethod can be used to treat a degenerative disorder of a knee, such aswhich might be the result of traumatic injury (e.g. a sports injury orexcessive wear) or osteoarthritis. The subject antagonists may beadministered as an injection into the joint with, for instance, anarthroscopic needle. In some instances, the injected agent can be in theform of a hydrogel or other slow release vehicle described above inorder to permit a more extended and regular contact of the agent withthe treated tissue.

The present invention further contemplates the use of the subject methodin the field of cartilage transplantation and prosthetic devicetherapies. However, problems arise, for instance, because thecharacteristics of cartilage and fibrocartilage varies between differenttissue: such as between articular, meniscal cartilage, ligaments, andtendons, between the two ends of the same ligament or tendon, andbetween the superficial and deep parts of the tissue. The zonalarrangement of these tissues may reflect a gradual change in mechanicalproperties, and failure occurs when implanted tissue, which has notdifferentiated under those conditions, lacks the ability toappropriately respond. For instance, when meniscal cartilage is used torepair anterior cruciate ligaments, the tissue undergoes a metaplasia topure fibrous tissue. By regulating the rate of chondrogenesis, thesubject method can be used to particularly address this problem, byhelping to adaptively control the implanted cells in the new environmentand effectively resemble hypertrophic chondrocytes of an earlierdevelopmental stage of the tissue.

In similar fashion, the subject method can be applied to enhancing boththe generation of prosthetic cartilage devices and to theirimplantation. The need for improved treatment has motivated researchaimed at creating new cartilage that is based oncollagen-glycosaminoglycan templates (Stone et al. (1990) Clin OrthopRelat Red 252:129), isolated chondrocytes (Grande et al. (1989) J OrthopRes 7:208; and Takigawa et al. (1987) Bone Miner 2:449), andchondrocytes attached to natural or synthetic polymers (Walitani et al.(1989) J Bone Jt Surg 71B; 74; Vacanti et al. (1991) Plast Reconstr Surg88:753; von Schroeder et al. (1991) J Biomed Mater Res 25:329; Freed etal. (1993) J Biomed Mater Res 27:11; and the Vacanti et al. U.S. Pat.No. 5,041,138). For example, chondrocytes can be grown in culture onbiodegradable, biocompatible highly porous scaffolds formed frompolymers such as polyglycolic acid, polylactic acid, agarose gel, orother polymers which degrade over time as function of hydrolysis of thepolymer backbone into innocuous monomers. The matrices are designed toallow adequate nutrient and gas exchange to the cells until engraftmentoccurs. The cells can be cultured in vitro until adequate cell volumeand density has developed for the cells to be implanted. One advantageof the matrices is that they can be cast or molded into a desired shapeon an individual basis, so that the final product closely resembles thepatient's own car or nose (by way of example), or flexible matrices canbe used which allow for manipulation at the time of implantation, as ina joint.

In one embodiment of the subject method, the implants are contacted witha hedgehog antagonist during certain stages of the culturing process inorder to manage the rate of differentiation of chondrocytes and theformation of hypertrophic chrondrocytes in the culture.

In another embodiment, the implanted device is treated with a hedgehogagonist in order to actively remodel the implanted matrix and to make itmore suitable for its intended function. As set out above with respectto tissue transplants, the artificial transplants suffer from the samedeficiency of not being derived in a setting which is comparable to theactual mechanical environment in which the matrix is implanted. Theability to regulate the chondrocytes in the matrix by the subject methodcan allow the implant to acquire characteristics similar to the tissuefor which it is intended to replace.

In yet another embodiment, the subject method is used to enhanceattachment of prosthetic devices. To illustrate, the subject method canbe used in the implantation of a periodontal prosthesis, wherein thetreatment of the surrounding connective tissue stimulates formation ofperiodontal ligament about the prosthesis.

In still further embodiments, the subject method can be employed as partof a regimen for the generation of bone (osteogenesis) at a site in theanimal where such skeletal tissue is deficient. Indian hedgehog isparticularly associated with the hypertrophic chondrocytes that areultimately replaced by osteoblasts. For instance, administration of ahedgehog antagonists of the present invention can be employed as part ofa method for regulating the rate of bone loss in a subject. For example,preparations comprising hedgehog antagonists can be employed, forexample, to control endochondral ossification in the formation of a“model” for ossification.

In yet another embodiment of the present invention, a hedgehogantagonist can be used to regulate spermatogenesis. The hedgehogproteins, particularly Dhh, have been shown to be involved in thedifferentiation and/or proliferation and maintenance of testicular germcells. Dhh expression is initiated in Sertoli cell precursors shortlyafter the activation of Sry and persists in the testis into the adult.Males are viable but infertile, owing to a complete absence of maturesperm. Examination of the developing testis in different geneticbackgrounds suggests that Dhh regulates both early and late stages ofspermatogenesis. Bitgood et al. (1996) Curr Biol 6:298. The subjectmethod can be utilized to block the action of a naturally-occurringhedgehog protein. In a preferred embodiment, the hedgehog antagonistinhibits the biological activity of Desert hedgehog with respect tospermatogenesis, and can be used as a contraceptive. In similar fashion,hedgehog antagonists of the subject method are potentially useful formodulating normal ovarian function.

The subject method also has wide applicability to the treatment orprophylaxis of disorders afflicting epithelial tissue, as well as incosmetic uses. In general, the method can be characterized as includinga step of administering to an animal an amount of a hedgehog antagonisteffective to alter the growth state of a treated epithelial tissue. Themode of administration and dosage regimens will vary depending on theepithelial tissue(s) which is to be treated. For example, topicalformulations will be preferred where the treated tissue is epidermaltissue, such as dermal or mucosal tissues.

A method which “promotes the healing of a wound” results in the woundhealing more quickly as a result of the treatment than a similar woundheals in the absence of the treatment. “Promotion of wound healing” canalso mean that the method regulates the proliferation and/or growth of,inter alia, keratinocytes, or that the wound heals with less scarring,less wound contraction, less collagen deposition and more superficialsurface area. In certain instances, “promotion of wound healing” canalso mean that certain methods of wound healing have improved successrates, (e.g. the take rates of skin grafts,) when used together with themethod of the present invention.

Despite significant progress in reconstructive surgical techniques,scarring can be an important obstacle in regaining normal function andappearance of healed skin. This is particularly true when pathologicscarring such as keloids or hypertrophic scars of the hands or facecauses functional disability or physical deformity. In the severestcircumstances, such scarring may precipitate psychosocial distress and alife of economic deprivation. Wound repair includes the stages ofhemostasis, inflammation, proliferation, and remodeling. Theproliferative stage involves multiplication of fibroblasts andendothelial and epithelial cells. Through the use of the subject method,the rate of proliferation of epithelial cells in and proximal to thewound can be controlled in order to accelerate closure of the woundand/or minimize the formation of scar tissue.

The present treatment can also be effective as part of a therapeuticregimen for treating oral and paraoral ulcers, e.g. resulting fromradiation and/or chemotherapy. Such ulcers commonly develop within daysafter chemotherapy or radiation therapy. These ulcers usually begin assmall, painful irregularly shaped lesions usually covered by a delicategray necrotic membrane and surrounded by inflammatory tissue. In manyinstances, lack of treatment results in proliferation of tissue aroundthe periphery of the lesion on an inflammatory basis. For instance, theepithelium bordering the ulcer usually demonstrates proliferativeactivity, resulting in loss of continuity of surface epithelium. Theselesions, because of their size and loss of epithelial integrity, disposethe body to potential secondary infection. Routine ingestion of food andwater becomes a very painful event and, if the ulcers proliferatethroughout the alimentary canal, diarrhea usually is evident with allits complicating factors. According to the present invention, atreatment for such ulcers which includes application of an hedgehogantagonist can reduce the abnormal proliferation and differentiation ofthe affected epithelium, helping to reduce the severity of subsequentinflammatory events.

The subject method and compositions can also be used to treat woundsresulting from dermatological diseases, such as lesions resulting fromautoimmune disorders such as psoriasis. Atopic dermititis refers to skintrauma resulting from allergies associated with an immune responsecaused by allergens such as pollens, foods, dander, insect venoms andplant toxins.

In other embodiments, antiproliferative preparations of hedgehogantagonists can be used to inhibit lens epithelial cell proliferation toprevent post-operative complications of extracapsular cataractextraction. Cataract is an intractable eye disease and various studieson a treatment of cataract have been made. But at present, the treatmentof cataract is attained by surgical operations. Cataract surgery hasbeen applied for a long time and various operative methods have beenexamined. Extracapsular lens extraction has become the method of choicefor removing cataracts. The major medical advantages of this techniqueover intracapsular extraction are lower incidence of aphakic cystoidmacular edema and retinal detachment. Extracapsular extraction is alsorequired for implantation of posterior chamber type intraocular lenseswhich are now considered to be the lenses of choice in most cases.

However, a disadvantage of extracapsular cataract extraction is the highincidence of posterior lens capsule opacification, often calledafter-cataract, which can occur in up to 50% of cases within three yearsafter surgery. After-cataract is caused by proliferation of equatorialand anterior capsule lens epithelial cells which remain afterextracapsular lens extraction. These cells proliferate to causeSommerling rings, and along with fibroblasts which also deposit andoccur on the posterior capsule, cause opacification of the posteriorcapsule, which interferes with vision. Prevention of after-cataractwould be preferable to treatment. To inhibit secondary cataractformation, the subject method provides a means for inhibitingproliferation of the remaining lens epithelial cells. For example, suchcells can be induced to remain quiescent by instilling a solutioncontaining an hedgehog antagonist preparation into the anterior chamberof the eye after lens removal. Furthermore, the solution can beosmotically balanced to provide minimal effective dosage when instilledinto the anterior chamber of the eye, thereby inhibiting subcapsularepithelial growth with some specificity.

The subject method can also be used in the treatment of comeopathiesmarked by corneal epithelial cell proliferation, as for example inocular epithelial disorders such as epithelial downgrowth or squamouscell carcinomas of the ocular surface.

Levine et al. (1997) J Neurosci 17:6277 show that hedgehog proteins canregulate mitogenesis and photoreceptor differentiation in the vertebrateretina, and Ihh is a candidate factor from the pigmented epithelium topromote retinal progenitor proliferation and photoreceptordifferentiation. Likewise, Jensen et al. (1997) Development 124:363demonstrated that treatment of cultures of perinatal mouse retinal cellswith the amino-terminal fragment of Sonic hedgehog protein results in anincrease in the proportion of cells that incorporate bromodeoxuridine,in total cell numbers, and in rod photoreceptors, amacrine cells andMuller glial cells, suggesting that Sonic hedgehog promotes theproliferation of retinal precursor cells. Thus the subject method can beused in the treatment of proliferative diseases of retinal cells andregulate photoreceptor differentiation.

Yet another aspect of the present invention relates to the use of thesubject method to control hair growth. Hair is basically composed ofkeratin, a tough and insoluble protein; its chief strength lies in itsdisulphide bond of cystine. Each individual hair comprises a cylindricalshaft and a root, and is contained in a follicle, a flask-likedepression in the skin. The bottom of the follicle contains afinger-like projection termed the papilla, which consists of connectivetissue from which hair grows, and through which blood vessels supply thecells with nourishment. The shaft is the part that extends outwards fromthe skin surface, whilst the root has been described as the buried partof the hair. The base of the root expands into the hair bulb, whichrests upon the papilla. Cells from which the hair is produced grow inthe bulb of the follicle; they are extruded in the form of fibers as thecells proliferate in the follicle. Hair “growth” refers to the formationand elongation of the hair fiber by the dividing cells.

As is well knows in the art, the common hair cycle is divided into threestages: anagen, catagen and telogen. During the active phase (anagen),the epidermal stem cells of the dermal papilla divide rapidly. Daughtercells move upward and differentiate to form the concentric layers of thehair itself. The transitional stage, catagen, is marked by the cessationof mitosis of the stem cells in the follicle. The resting stage is knownas telogen, where the hair is retained within the scalp for severalweeks before an emerging new hair developing below it dislodges thetelogen-phase shaft from its follicle. From this model it has becomeclear that the larger the pool of dividing stem cells that differentiateinto hair cells the more hair growth occurs. Accordingly, methods forincreasing or reducing hair growth can be carried out by potentiating orinhibiting, respectively, the proliferation of these stem cells.

In certain embodimemts, the subject method can be employed as a way ofreducing the growth of human hair as opposed to its conventional removalby cutting, shaving, or depilation. For instance, the present method canbe used in the treatment of trichosis characterized by abnormally rapidor dense growth of hair, e.g. hypertrichosis. In an exemplaryembodiment, hedgehog antagonists can be used to manage hirsutism, adisorder marked by abnormal hairiness. The subject method can alsoprovide a process for extending the duration of depilation.

Moreover, because a hedgehog antagonist will often be cytostatic toepithelial cells, rather than cytotoxic, such agents can be used toprotect hair follicle cells from cytotoxic agents which requireprogression into S-phase of the cell-cycle for efficacy, e.g.radiation-induced death. Treatment by the subject method can provideprotection by causing the hair follicle cells to become quiescent, e.g.,by inhibiting the cells from entering S phase, and thereby preventingthe follicle cells from undergoing mitotic catastrophe or programmedcell death. For instance, hedgehog antagonists can be used for patientsundergoing chemo- or radiation-therapies which ordinarily result in hairloss. By inhibiting cell-cycle progression during such therapies, thesubject treatment can protect hair follicle cells from death which mightotherwise result from activation of cell death programs. After thetherapy has concluded, the instant method can also be removed withconcommitant relief of the inhibition of follicle cell proliferation.

The subject method can also be used in the treatment of folliculitis,such as folliculitis decalvans, folliculitis ulerythematosa reticulataor keloid folliculitis. For example, a cosmetic prepration of anhedgehog antagonist can be applied topically in the treatment ofpseudofolliculitis, a chronic disorder occurring most often in thesubmandibular region of the neck and associated with shaving, thecharacteristic lesions of which are erythematous papules and pustulescontaining buried hairs.

In another aspect of the invention, the subject method can be used toinduce differentiation of epithelially-derived tissue. Such forms ofthese molecules can provide a basis for differentiation therapy for thetreatment of hyperplastic and/or neoplastic conditions involvingepithelial tissue. For example, such preparations can be used for thetreatment of cutaneous diseases in which there is abnormal proliferationor growth of cells of the skin.

For instance, the pharmaceutical preparations of the invention areintended for the treatment of hyperplastic epidermal conditions, such askeratosis, as well as for the treatment of neoplastic epidermalconditions such as those characterized by a high proliferation rate forvarious skin cancers, as for example basal cell carcinoma or squamouscell carcinoma. The subject method can also be used in the treatment ofautoimmune diseases affecting the skin, in particular, of dermatologicaldiseases involving morbid proliferation and/or keratinization of theepidermis, as for example, caused by psoriasis or atopic dermatosis.

Many common diseases of the skin, such as psoriasis squamous cellcarcinoma, keratoacanthoma and actinic keratosis are characterized bylocalized abnormal proliferation and growth. For example, in psoriasis,which is characterized by scaly, red, elevated plaques on the skin, thekeratinocytes are known to proliferate much more rapidly than normal andto differentiate less completely.

In one embodiment, the preparations of the present invention aresuitable for the treatment of dermatological ailments linked tokeratinization disorders causing abnormal proliferation of skin cells,which disorders may be marked by either inflammatory or non-inflammatorycomponents. To illustrate, therapeutic preparations of a hedgehogantagonist, e.g., which promotes quiescense or differentiation can beused to treat varying forms of psoriasis, be they cutaneous, mucosal orungual. Psoriasis, as described above, is typically characterized byepidermal keratinocytes which display marked proliferative activationand differentiation along a “regenerative” pathway. Treatment with anantiproliferative embodiment of the subject method can be used toreverse the pathological epidermal activiation and can provide a basisfor sustained remission of the disease.

A variety of other keratotic lesions are also candidates for treatmentwith the subject method. Actinic keratoses, for example, are superficialinflammatory premalignant tumors arising on sun-exposed and irradiatedskin. The lesions are erythematous to brown with variable scaling.Current therapies include excisional and cryosurgery. These treatmentsare painful, however, and often produce cosmetically unacceptablescarring. Accordingly, treatment of keratosis, such as actinickeratosis, can include application, preferably topical, of a hedgehogantagonist composition in amounts sufficient to inhibithyperproliferation of epidermal/epidermoid cells of the lesion.

Acne represents yet another dermatologic ailment which may be treated bythe subject method. Acne vulgaris, for instance, is a multifactorialdisease most commonly occurring in teenagers and young adults, and ischaracterized by the appearance of inflammatory and noninflammatorylesions on the face and upper trunk. The basic defect which gives riseto acne vulgaris is hypercornification of the duct of a hyperactivesebaceous gland. Hypercornification blocks the normal mobility of skinand follicle microorganisms, and in so doing, stimulates the release oflipases by Propinobacterium acnes and Staphylococcits epidermidisbacteria and Pitrosporum ovale, a yeast. Treatment with anantiproliferative hedgehog antagonist, particularly topicalpreparations, may be useful for preventing the transitional features ofthe ducts, e.g. hypercomification, which lead to lesion formation. Thesubject treatment may further include, for example, antibiotics,retinoids and antiandrogens.

The present invention also provides a method for treating various formsof dermatitis. Dermatitis is a descriptive term referring to poorlydemarcated lesions which are either pruritic, erythematous, scaley,blistered, weeping, fissured or crusted. These lesions arise from any ofa wide variety of causes. The most common types of dermatitis areatopic, contact and diaper dermatitis. For instance, seborrheicdermatitis is a chronic, usually pruritic, dermatitis with erythema,dry, moist, or greasy scaling, and yellow crusted patches on variousareas, especially the scalp, with exfoliation of an excessive amount ofdry scales. The subject method can also be used in the treatment ofstasis dermatitis, an often chronic, usually eczematous dermatitis.Actinic dermatitis is dermatitis that due to exposure to actinicradiation such as that from the sun, ultraviolet waves or x- orgamma-radiation. According to the present invention, the subject methodcan be used in the treatment and/or prevention of certain symptoms ofdermatitis caused by unwanted proliferation of epithelial cells. Suchtherapies for these various forms of dermatitis can also include topicaland systemic corticosteroids, antipurities, and antibiotics.

Ailments which may be treated by the subject method are disordersspecific to non-humans, such as mange.

In still another embodiment, the subject method can be used in thetreatment of human cancers, particularly basal cell carcinomas and othertumors of epithelial tissues such as the skin. For example, hedgehogantagonists can be employed, in the subject method, as part of atreatment for basal cell nevus syndrome (BCNS), and other other humancarcinomas, adenocarcinomas, sarcomas and the like.

In one aspect, the present invention provides pharmaceuticalpreparations and methods for controlling the formation ofmegakaryocyte-derived cells and/or controlling the functionalperformance of megakaryocyte-derived cells. For instance, certain of thecompositions disclosed herein may be applied to the treatment orprevention of a variety hyperplastic or neoplastic conditions affectingplatelets.

The hedgehog antagonists for use in the subject method may beconveniently formulated for administration with a biologicallyacceptable medium, such as water, buffered saline, polyol (for example,glycerol, propylene glycol, liquid polyethylene glycol and the like) orsuitable mixtures thereof. The optimum concentration of the activeingredient(s) in the chosen medium can be determined empirically,according to procedures well known to medicinal chemists. As usedherein, “biologically acceptable medium” includes any and all solvents,dispersion media, and the like which may be appropriate for the desiredroute of administration of the pharmaceutical preparation. The use ofsuch media for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe activity of the hedgehog antagonist, its use in the pharmaceuticalpreparation of the invention is contemplated. Suitable vehicles andtheir formulation inclusive of other proteins are described, forexample, in the book Remington's Pharmaceutical Sciences (Remington'sPharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA1985). These vehicles include injectable “deposit formulations”.

Pharmaceutical formulations of the present invention can also includeveterinary compositions, e.g. pharmaceutical preparations of thehedgehog antagonists suitable for veterinary uses, e.g., for thetreatment of live stock or domestic animals, e.g., dogs.

Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinacious biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a hedgehog antagonist at aparticular target site.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral and topical administrations are preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration.” “administered systemically.”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms such as described below orby other conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular hedegehog antagonist employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisinvention for a patient will range from about 0.0001 to about 100 mg perkilogram of body weight per day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable carriers and can also beadministered in conjunction with other antimicrobial agents such aspenicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy, thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticaleffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

V. Pharmaceutical Compositions

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition). The hedgehog antagonistsaccording to the invention may be formulated for administration in anyconvenient way for use in human or veterinary medicine.

Thus, another aspect of the present invention provides pharmaceuticallyacceptable compositions comprising a therapeutically-effective amount ofone or more of the compounds described above, formulated together withone or more pharmaceutically acceptable carriers (additives) and/ordiluents. As described in detail below, the pharmaceutical compositionsof the present invention may be specially formulated for administrationin solid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), tablets, boluses, powders, granules, pastesfor application to the tongue; (2) parenteral administration, forexample, by subcutaneous, intramuscular or intravenous injection as, forexample, a sterile solution or suspension; (3) topical application, forexample, as a cream, ointment or spray applied to the skin; or (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam. However, in certain embodiments the subject compounds may besimply dissolved or suspended in sterile water.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect by inhibiting a hedgehog signaling pathway in atleast a sub-population of cells in an animal and thereby blocking thebiological consequences of that pathway in the treated cells, at areasonable benefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject antagonistsfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

As set out above, certain embodiments of the present hedgehogantagonists may contain a basic functional group, such as amino oralkylamino, and are, thus, capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptableacids. The term “pharmaceutically-acceptable salts” in this respect,refers to the relatively non-toxic, inorganic and organic acid additionsalts of compounds of the present invention. These salts can be preparedin situ during the final isolation and purification of the compounds ofthe invention, or by separately reacting a purified compound of theinvention in its free base form with a suitable organic or inorganicacid, and isolating the salt thus formed. Representative salts includethe hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate,acetate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, napthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts and the like. (See, for example, Berge et al.(1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19)

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. The term “pharmaceutically-acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ during the final isolation and purification of thecompounds, or by separately reacting the purified compound in its freeacid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically-acceptable metal cation, with ammonia,or with a pharmaceutically-acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like. (See, forexample, Berge et al. supra).

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like: (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about1 percent to about ninety-nine percent of active ingredient, preferablyfrom about 5 percent to about 70 percent, most preferably from about 10percent to about 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically-acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

It is known that sterols, such as cholesterol, will form complexes withcyclodextrins. Thus, in preferred embodiments, where the inhibitor is asteroidal alkaloid, it may be formulated with cyclodextrins, such as α-,β- and γ-cyclodextrin, dimethyl-β cyclodextrin and2-hydroxypropyl-β-cyclodextrin.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active hedgehog antagonist.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the hedgehog antagonistsin the proper medium. Absorption enhancers can also be used to increasethe flux of the hedgehog antagonists across the skin. The rate of suchflux can be controlled by either providing a rate controlling membraneor dispersing the compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed. The way in whichsuch feed premixes and complete rations can be prepared and administeredare described in reference books (such as “Applied Animal Nutrition”.W.H. Freedman and CO., San Francisco, U.S.A., 1969 or “Livestock Feedsand Feeding” O and B books. Corvallis, Oreg., U.S.A., 1977).

VI. Synthetic Schemes and Identification of Active Antagonists

The subjects steroidal alkaloids, and congeners thereof, can be preparedreadily by employing the cross-coupling technologies of Suzuki, Stille,and the like. These coupling reactions are carried out under relativelymild conditions and tolerate a wide range of “spectator” functionality.

a. Combinatorial Libraries

The compounds of the present invention, particularly libraries ofvariants having various representative classes of substituents, areamenable to combinatorial chemistry and other parallel synthesis schemes(see, for example, PCT WO 94/08051). The result is that large librariesof related compounds, e.g. a variegated library of compounds representedabove, can be screened rapidly in high throughput assays in order toidentify potential hedgehog antagonists lead compounds, as well as torefine the specificity, toxicity, and/or cytotoxic-kinetic profile of alead compound. For instance, hedgehog bioactivity assays as describedabove can be used to screen a library of the subject compounds for thosehaving antagonist activity toward all or a particular hedgehog isoformor activity.

Simply for illustration, a combinatorial library for the purposes of thepresent invention is a mixture of chemically related compounds which maybe screened together for a desired property. The preparation of manyrelated compounds in a single reaction greatly reduces and simplifiesthe number of screening processes which need to be carried out.Screening for the appropriate physical properties can be done byconventional methods.

Diversity in the library can be created at a variety of differentlevels. For instance, the substrate aryl groups used in thecombinatorial reactions can be diverse in terms of the core aryl moiety,e.g. a variegation in terms of the ring structure, and/or can be variedwith respect to the other substituents.

A variety of techniques are available in the art for generatingcombinatorial libraries of small organic molecules such as the subjecthedgehog antagonists. See, for example, Blondelle et al. (1995) TrendsAnal. Chem. 14:83; the Affymax U.S. Pat. Nos. 5,359,115 and 5,362,899;the Ellman U.S. Pat. No. 5,288,514; the Still et al. PCT publication WO94/08051; Chen et al. (1994) JACS 116:2661; Kerr et al. (1993) JACS115:252; PCT publications WO92/10092, WO93/09668 and WO91/07087; and theLerner et al. PCT publication WO93/20242). Accordingly, a variety oflibraries on the order of about 100 to 1,000,000 or more diversomers ofthe subject hedgehog antagonists can be synthesized and screened forparticular activity or property.

In an exemplary embodiment, a library of candidate hedgehog antagonistsdiversomers can be synthesized utilizing a scheme adapted to thetechniques described in the Still et al. PCT publication WO 94/08051,e.g., being linked to a polymer bead by a hydrolyzable or photolyzablegroup e.g., located at one of the positions of the candidate antagonistsor a substituent of a synthetic intermediate. According to the Still etal. technique, the library is synthesized on a set of beads, each beadincluding a set of tags identifying the particular diversomer on thatbead. The bead library can then be “plated” on a lawn ofhedgehog-sensitive cells for which an inhibitor is sought. Thediversomers can be released from the bead, e.g. by hydrolysis. Beadssurrounded by areas of no, or diminished, hedgehog sensitivity (e.g., toexogeneously added hedgehog protein), e.g. a “halo”, can be selected,and their tags can be “read” to establish the identity of the particulardiversomer.

b. Screening Assays

There are a variety of assays availble for determining the ability of acompound to inhibit hedgehog-mediated signaling, many of which can bedisposed in high throughput formats. In many drug screening programswhich test libraries of compounds and natural extracts, high throughputassays are desirable in order to maximize the number of compoundssurveyed in a given period of time. Thus, libraries of synthetic andnatural products can be sampled for other steroidal and non-steroidalcompounds which have similar activity to jervine with respect inhibitionof hedgehog signals.

The availability of purified and recombinant hedgehog polypeptidesfacilitates the generation of assay systems which can be used to screenfor drugs, such as small organic molecules, which are antagonists of thenormal cellular function of a hedgehog, particularly its role in thepathogenesis of cell proliferation and/or differentiation. In oneembodiment, the assay evaluates the ability of a compound to modulatebinding between a hedgehog polypeptide and a hedgehog receptor such aspatched. In other embodiments, the assay merely scores for the abilityof a test compound to alter hedgehog-mediated signal transduction. Inthis manner, a variety of antagonists can be identified. A variety ofassay formats will suffice and, in light of the present disclosure, willbe comprehended by skilled artisan.

Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity with receptorproteins.

While not wishing to be bound by any particular theory, should jervineand other steroidal alkaloids exert their activity in the hedgehogsignal pathway by interfering with cholesterol-derived hedgehog, e.g.,through interaction with the sterol sensing domain of patched (see, e.g.Loftus et al. (1997) Science 277:232), an exemplary screening assay fora hedgehog antagonist comprises contacting a compound of interest with amixture including a hedgehog receptor protein (e.g., a cell expressingthe patched receptor) and a hedgehog protein under conditions in whichthe receptor is ordinarily capable of binding the hedgehog protein, orat least jervine. To the mixture is then added a composition containinga test compound. Detection and quantification of receptor/hedgehogand/or receptor/jervine complexes provides a means for determining thetest compound's efficacy at inhibiting (or potentiating) complexformation between the receptor protein and the hedgehog polypeptide orthe jervine antagonist. The efficacy of the compound can be assessed bygenerating dose response curves from data obtained using variousconcentrations of the test compound, and by comparing the results tothat obtained with jervine. Moreover, a control assay can also beperformed to provide a baseline for comparison. In the control assay,isolated and purified hedgehog polypeptide is added to the receptorprotein, and the formation of receptor/hedgehog complex is quantitatedin the absence of the test compound.

In an illustrative embodiment, the polypeptide utilized as a hedgehogreceptor can be generated from the patched protein, and in particular,includes the steroid sensing domain, e.g. and a soluble portion of theprotein including a functional steroid sensing domain. Accordingly, anexemplary screening assay includes all or a suitable portion of thepatched protein which can be obtained from, for example, the humanpatched gene (GenBank U43148) or other vertebrate sources (see GenBankAccession numbers U40074 for chicken patched and U46155 for mousepatched), as well as from drosophila (GenBank Accession number M28999)or other invertebrate sources. The patched protein can be provided inthe screening assay as a whole protein (preferably expressed on thesurface of a cell), or alternatively as a fragment of the full lengthprotein, e.g. which includes the steroid sensing domain and/or at leasta portion which binds to hedgehog polypeptides, e.g. as one or both ofthe substantial extracellular domains (e.g. corresponding to residuesAsn120-Ser438 and/or Arg770-Trp1027 of the human patched protein. Forinstance, the patched protein can be provided in soluble form, as forexample a preparation of one of the extracellular domains, or apreparation of both of the extracellular domains which are covalentlyconnected by an unstructured linker (see, for example, Huston et al.(1988) PNAS 85:4879; and U.S. Pat. No. 5,091,513). In other embodiments,the protein can be provided as part of a liposomal preparation orexpressed on the surface of a cell. The patched protein can derived froma recombinant gene, e.g., being ectopically expressed in a heterologouscell. For instance, the protein can be expressed on oocytes, mammaliancells (e.g., COS, CHO, 3T3 or the like), or yeast cell by standardrecombinant DNA techniques. These recombinant cells can be used forreceptor binding, signal transduction or gene expression assays. Stoneet al. (1996) Nature 384:129-34; and Marigo et al. (1996) Nature384:176-9 illustrate binding assays of human hedgehog to patched, suchas a chicken patched protein ectopically expressed in Xenopus laevisoocytes. The assay system of Marigo et al. for example, can be adaptedto the present drug screening assays. As illustrated in that reference,Shh binds to the patched protein in a selective, saturable,dose-dependent manner, thus demonstrating that patched is a receptor forShh.

Complex formation between the hedgehog polypeptide or jervine and ahedgehog receptor may be detected by a variety of techniques. Forinstance, modulation of the formation of complexes can be quantitatedusing, for example, detectably labelled proteins such as radiolabelled,fluorescently labelled, or enzymatically labelled hedgehog polypeptides,by immunoassay, or by chromatographic detection.

Typically, for cell-free assays, it will be desirable to immobilizeeither the hedgehog receptor or the hedgehog polypeptide or jervinemolecule to facilitate separation of receptor complexes from uncomplexedforms of one of the protein, as well as to accommodate automation of theassay. In one embodiment, a fusion protein can be provided which adds adomain that allows the protein to be bound to a matrix. For example,glutathione-S-transferase/receptor (GST/receptor) fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with jervine or the hedgehog polypeptide, e.g. an ³⁵S-labeledhedgehog polypeptide, and the test compound and incubated underconditions conducive to complex formation, e.g. at physiologicalconditions for salt and pH, though slightly more stringent conditionsmay be desired. Following incubation, the beads are washed to remove anyunbound ligand, and the matrix bead-bound radiolabel determined directly(e.g. beads placed in scintillant), or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the bead, separated by SDS-PAGE gel, and the level ofhedgehog polypeptide or jervine found in the bead fraction quantitatedfrom the gel using standard techniques (HPLC, gel electrophoresis, etc).

Where the desired portion of the hedgehog receptor (or other hedgehogbinding molecule) cannot be provided in soluble form, liposomal vesiclescan be used to provide manipulatable and isolatable sources of thereceptor. For example, both authentic and recombinant forms of thepatched protein can be reconstituted in artificial lipid vesicles (e.g.phosphatidylcholine liposomes) or in cell membrane-derived vesicles(see, for example. Bear et al. (1992) Cell 68:809-818; Newton et al.(1983) Biochemistry 22:6110-6117; and Reber et al. (1987) J Biol Chem262:11369-11374).

In addition to cell-free assays, such as described above, the compoundsof the subject invention can also be tested in cell-based assays. In oneembodiment, cell which are sensitive to hedgehog induction, e.g.patched-expressing cells or other cells sensitive to hedgehog induction,can be contacted with a hedgehog protein and a test agent of interest,with the assay scoring for anything from simple binding to the cell toinhibition in hedgehog inductive responses by the target cell in thepresence and absence of the test agent.

In addition to characterizing cells that naturally express the patchedprotein, cells which have been genetically engineered to ectopicallyexpress patched can be utilized for drug screening assays. As anexample, cells which either express low levels or lack expression of thepatched protein, e.g. Xenopus laevis oocytes, COS cells or yeast cells,can be genetically modified using standard techniques to ectopicallyexpress the patched protein. (see Marigo et al., supra).

The resulting recombinant cells, e.g., which express a functionalpatched receptor, can be utilized in receptor binding assays to identifyagonist or anatagonsts of hedgehog binding. Binding assays can beperformed using whole cells. Furthermore, the recombinant cells of thepresent invention can be engineered to include other heterolgous genesencoding proteins involved in hedgehog-dependent siganl pathways. Forexample, the gene products of one or more of smoothened, costal-2,fused, and/or suppressor of fused can be co-expressed with patched inthe reagent cell, with assays being sensitive to the functionalreconstituion of the hedgehog signal transduction cascade.

Alternatively, liposomal preparations using reconstituted patchedprotein can be utilized. Patched protein purified from detergentextracts from both authentic and recombinant origins can bereconstituted in in artificial lipid vesicles (e.g. phosphatidylcholineliposomes) or in cell membrane-derived vesicles (see, for example, Bearet al. (1992) Cell 68:809-818; Newton et al. (1983) Biochemistry22:6110-6117; and Reber et al. (1987) J Biol Chem 262:11369-11374). Thelamellar structure and size of the resulting liposomes can becharacterized using electron microscopy. External orientation of thepatched protein in the reconstituted membranes can be demonstrated, forexample, by immunoelectron microscopy. The hedgehog protein bindingactivity of liposomes containing patched and liposomes without theprotein in the presence of candidate agents can be compared in order toidentify potential modulators of the hedgehog-patched interaction.

The hedgehog protein used in these cell-based assays can be provided asa purified source (natural or recombinant in origin), or in the form ofcells/tissue which express the protein and which are co-cultured withthe target cells, and is preferably a cholesterol-derived form. Inaddition to binding studies, by detecting changes in intracellularsignals, such as alterations in second messengers or gene expression, inpatched-expressing cells contacted with a test agent, candidate hedgehogantagonists can be identified.

A number of gene products have been implicated in patched-mediatedsignal transduction, including patched, the transcription factor cubitusinterruptus (ci), the serine/threonine kinase fused (fu) and the geneproducts of costal-2, smoothened and suppressor of fused.

The induction of cells by hedgehog proteins sets in motion a cascadeinvolving the activation and inhibition of downstream effectors, theultimate consequence of which is, in some instances, a detectable changein the transcription or translation of a gene, Potential transcriptionaltargets of hedgehog-mediated signaling are the patched gene (Hidalgo andIngham, 1990 Development 110, 291-301; Marigo et al., 1996) and thevertebrate homologs of the drosophila cubitus interruptus gene, the GLIgenes (Hui et al. (1994) Dev Biol 162:402-413). Patched gene expressionhas been shown to be induced in cells of the limb bud and the neuralplate that are responsive to Shh. (Marigo et al. (1996) PNAS 93:9346-51;Marigo et al. (1996) Development 122:1225-1233). The GLI genes encodeputative transcription factors having zinc finger DNA binding domains(Orenic et al. (1990) Genes & Dev 4:1053-1067; Kinzler et al. (1990) MolCell Biol 10:634-642). Transcription of the GLI gene has been reportedto be upregulated in response to hedgehog in limb buds, whiletranscription of the GLI3 gene is downregulated in response to hedgehoginduction (Marigo et al. (1996) Development 122:1225-1233). By selectingtranscriptional regulatory sequences from such target genes, e.g. frompatched or GLI genes, that are responsible for the up- or downregulation of these genes in response to hedgehog signalling, andoperatively linking such promoters to a reporter gene, one can derive atranscription based assay which is sensitive to the ability of aspecific test compound to modify hedgehog-mediated signalling pathways.Expression of the reporter gene, thus, provides a valuable screeningtool for the development of compounds that act as antagonists ofhedgehog.

Reporter gene based assays of this invention measure the end stage ofthe above described cascade of events, e.g., transcriptional modulation.Accordingly, in practicing one embodiment of the assay, a reporter geneconstruct is inserted into the reagent cell in order to generate adetection signal dependent on hedgehog signaling. To identify potentialregulatory elements responsive to hedgehog signaling present in thetranscriptional regulatory sequence of a target gene, nested deletionsof genomic clones of the target gene can be constructed using standardtechniques. See, for example, Current Protocols in Molecular Biology,Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989); U.S.Pat. No. 5,266,488; Sato et al. (1995) J Biol Chem 270:10314-10322; andKube et al. (1995) Cytokine 7:1-7. A nested set of DNA fragments fromthe gene's 5′-flanking region are placed upstream of a reporter gene,such as the luciferase gene, and assayed for their ability to directreporter gene expression in patched expressing cells. Host cellstransiently transfected with reporter gene constructs can be scored forthe regulation of expression of the reporter gene in the presence andabsence of hedgehog to determine regulatory sequences which areresponsive to patched-dependent signalling.

In practicing one embodiment of the assay, a reporter gene construct isinserted into the reagent cell in order to generate a detection signaldependent on second messengers generated by induction with hedgehogprotein. Typically, the reporter gene construct will include a reportergene in operative linkage with one or more transcriptional regulatoryelements responsive to the hedgehog activity, with the level ofexpression of the reporter gene providing the hedgehog-dependentdetection signal. The amount of transcription from the reporter gene maybe measured using any method known to those of skill in the art to besuitable. For example, mRNA expression from the reporter gene may bedetected using RNAse protection or RNA-based PCR, or the protein productof the reporter gene may be identified by a characteristic stain or anintrinsic activity. The amount of expression from the reporter gene isthen compared to the amount of expression in either the same cell in theabsence of the test compound (or hedgehog) or it may be compared withthe amount of transcription in a substantially identical cell that lacksthe target receptor protein. Any statistically or otherwise significantdifference in the amount of transcription indicates that the testcompound has in some manner altered the signal transduction activity ofthe hedgehog protein, e.g., the test compound is a potential hedgehogantagonist.

As described in further detail below, in preferred embodiments the geneproduct of the reporter is detected by an intrinsic activity associatedwith that product. For instance, the reporter gene may encode a geneproduct that, by enzymatic activity, gives rise to a detection signalbased on color, fluorescence, or luminescence. In other preferredembodiments, the reporter or marker gene provides a selective growthadvantage, e.g., the reporter gene may enhance cell viability, relieve acell nutritional requirement, and/or provide resistance to a drug.

Preferred reporter genes are those that are readily detectable. Thereporter gene may also be included in the construct in the form of afusion gene with a gene that includes desired transcriptional regulatorysequences or exhibits other desirable properties. Examples of reportergenes include, but are not limited to CAT (chloramphenicol acetyltransferase) (Alton and Vapnek (1979), Nature 282: 864-869) luciferase,and other enzyme detection systems, such as beta-galactosidase, fireflyluciferase (deWet et al. (1987), Mol. Cell. Biol. 7:725-737); bacterialluciferase (Engebrecht and Silverman (1984), PNAS 1: 4154-4158; Baldwinet al. (1984), Biochemistry 23: 3663-3667); alkaline phosphatase (Toh etal. (1989) Eur. J. Biochem. 182: 231-238, Hall et al. (1983) J. Mol.Appl. Gen. 2: 101), human placental secreted alkaline phosphatase(Cullen and Malim (1992) Methods in Enzymol. 216:362-368).

Transcriptional control elements which may be included in a reportergene construct include, but are not limited to, promoters, enhancers,and repressor and activator binding sites. Suitable transcriptionalregulatory elements may be derived from the transcriptional regulatoryregions of genes whose expression is induced after modulation of ahedgehog signal transduction pathway. The characteristics of preferredgenes from which the transcriptional control elements are derivedinclude, but are not limited to, low or undetectable expression inquiescent cells, rapid induction at the transcriptional level withinminutes of extracellular simulation, induction that is transient andindependent of new protein synthesis, subsequent shut-off oftranscription requires new protein synthesis, and mRNAs transcribed fromthese genes have a short half-life. It is not necessary for all of theseproperties to be present.

Moreover, a number of assays are known in the art for detectinginhibitors of cholesterol biosynthesis and can be readily adapted fordetermining if the subject hedgehog antagonists disrupt cholesterolhomeosynthesis, e.g., by inhibiting biosynthesis and/or transport ofsterols.

To illustrate, pores formed in the membranes of animal cells bycomplexes of sterols and the polyene antibiotic amphotericin B canefficiently kill the cells. Thus, in the absence of exogenous sources ofcholesterol, inhibitors of enzymes in the cholesterol biosyntheticpathway render cells resistant to amphotericin B. However, in the caseof class 2 inhibitors, the increase in cholesterol in the plasmamembrane should in fact sensitive the cells to amphotericin B killing.Preincubation of Chinese hamster ovary cells with a test compound whichdisrupts cholesterol homoeostasis is a manner similar to jervine, suchas a test steroidal alkaloid, will sensitize cells to amphotericin Bkilling. This can be used, therefore, to assay test compounds and isamenable to high through-put screening. A simple two-step protocol inwhich cells are preincubated (15-24 h) with potential inhibitors andthen treated (3-6 h) with amphotericin B is described by Krieger (1983)Anal Biochem 135:383-391, and is a sensitive method for detecting direct(e.g., competitive) and regulatory inhibitors of cholesterolbiosynthesis. This protocol may prove useful in detecting potentialhedgehog antagonists.

SREBP cleavage-activating protein (SCAP) stimulates the proteolyticcleavage of membrane-bound SREBPs, thereby initiating the release ofNH2-terminal fragments from cell membranes. The liberated fragmentsenter the nucleus and stimulate transcription of genes involved insynthesis and uptake of cholesterol and fatty acids. Sterols represscleavage of SREBPs, apparently by interacting with the membraneattachment domain of SCAP. In one embodiment, the ability of a testagent to interfere with sterol transport in a manner similar to jervinecan be assayed by generation of activated SCAP or SREBP proteins. Forinstance, a particularly desirable embodiment, due to its ability to beused in high throughput screening, is a reporter gene based assay whichdetects SREBP-dependent gene transcription. A variey of genes have beendescribed in the art as including SREBP responsive elements, and whichare candidate for generation of reporter gene constructs. For example,Bist et al. (1997) PNAS 94 (20); 10693-8 describes the presence of SREBPresponsive elements in the Caveolin gene; Wang et al. (1997) J Biol Chem272:26367-74 describes such elements in the FAS gene; Magana et al.(1996) J Biol Chem 271:32689-94 describe the presence of SREBP-RE in thefatty-acid synthase gene; and Ericsson et al. (1996) PNAS 93:945-50describe SREBP binding sites in the farnesyl diphosphate synthase gene.Thus, such reporter gene constructs as the FAS promoter-luciferasereporter described by Wang et al. supra, or the squalene synthasepromoter-luciferase reporter described by Guan et al. (1997) J Biol Chem272:10295-302 can be utilized in an assay for detecting potentialequivalents to jervine. Briefly, in the absence of a class 2 inhibitor,sterol transport occurs at some level and SREBP-dependent transcriptionoccurs at a certain rate. Inhibition of sterol trafficking by jervine orthe like results in an increase in sterol precursors in the plasmamembrane and a decrease of such precrusors in the endoplasmic reticulum.The latter causes activation of SCAP-mediated cleavage of SREBP. and aconcomitant increase in expression of SREBP-RE reporter gene. Detectionof reporter gene expression can be accomplished by any of a wide rangeof techniques known in the art. For example, the reporter gene may onewhich confers drug resistance, such as to zeocin or hygromycin. Jervine,or another compound capable of inhibiting cholesterol homeostasis in asimilar manner, will cause increased resistance to the drug asexpression of the reporter gene is increased.

In still other embodiments, the ability of a test agent to effect theactivity of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductasecan be used to detect compounds which, like jervine, affect cholesterolhomeostasis. Conditions of low cholesterol or other sterols in theendoplasmic reticulum, such as caused by class 2 inhibitors likejervine, result in activation of HMG-CoA reductase. This activation canbe detected, for instance, by detecting increased expression of HMG-CoA(Chambers et al. (1997) Am J Med Genet 68:322-7) or by detectingincrease enzymatic activity, such as in the HMG-CoA reductase reductionof the substrate, [¹⁴C]HMG-CoA (see U.S. Pat. No. 5,753,675).

Exemplification

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

In order to demonstrate an effect on Shh signaling, we chose the chick(38) as a more tractable experimental system than the rodents, sheep andother mammals in which teratogen-induced HPE predominantly has beenstudied (14, 15, 16, 29, 39). Chick embryos are easily cultured andmanipulated and, as seen in FIG. 2, exposure of these embryos to jervineat the intermediate to definitive streak state (40) induced externalmalformations characteristic of HPE (similar results were obtained withcyclopamine; data not shown). The severity of these defects varied amongtreated embryos, as seen in panels B-E by the degree of loss of midlinestructures and approximation of paired lateral structures. These midlinedeficits thus result in the fusion of the mandibular and maxillaryprocesses as well as the optic vesicles and olfactory processes, withconsequent cyclopia and formation of a proboscis-like structureconsisting of fused nasal chambers in the most severely affected embryos(FIG. 2E).

As seen in FIG. 2 in ovo treatment produced variable defects and someembryos displayed normal morphology, even at the highest concentrationstested (50 μM, jervine 5/10 and cyclopamine 2/10, data now shown). Thevariability of these effects may be due to imprecise embryonic stagingand difficulties in applying these hydrophobic compounds uniformly. Toreduce this variability and better evaluate the potential effects ofteratogenic compounds on Shh signaling we established an explant assaythat allowed for precise tissue staging and more uniform application ofthe teratogen (41). As shown in FIG. 3A, medial neural plate withnotochord was explanted from a region just rostral to Hensen's node. Atthis level, the medial neural plte does not yet express floor plate cell(HNF3β) or motor neuron (Isl-1) markers (42, 43, data not shown),although the notochord does express Shh (44, 45, data not shown). Asseen in FIG. 3B, after a 40 hour incubation the neutral plate expressesHNF3β and Isl-1. Expression of these markers has been shown to dependupon Shh signaling, both in vivo and in vitro (2, 45), and these midlineexplants thus constitute an integrated assay of Shh signaling,comprising both inducing and target tissues.

To determine whether synthetic and plant-derived teratogens block Shhsignaling we exposed midline explants to varying concentrations of thedrugs AY 9944 and triparanol and to the steroidal alkaloids cyclopamineand jervine. As can be seen in FIGS. 3D-K, all of these compounds affectShh signaling, with a complete loss of HNF3 and Isl-1 expressionconsistently caused by sufficiently high concentrations (FIGS.3E,G,J,K). At concentrations several-fold below those required forcomplete inhibition, all of the teratogenic compounds are able to blockHNTF3β expression while retaining and often enhancing Isl-1 expression(FIGS. 3D,F,H,H). These effects are fully consistent with inhibition ofShh signaling (see below). In contract, the structurally related but notteratogenic steroidal alkaloid tomatidine (see FIG. 1, ref. 46, data notshown ) is unable to block expression of HNF3β and Isl-1, even atconcentrations two orders of magnitude higher than the inhibitoryconcentrations of jervine and cyclopamine (FIG. 3C).

Inhibitory Compounds do not Block Shh Processing

Because the midline explants contain both inducing and respondingtissues, we set out to distinguish possible effects of these inhibitorycompounds on signal production versus possible effects on signalresponse. The Shh protein undergoes an intramolecular processingreaction that involves internal cleavage and gives rise to anamino-terminal product (Shh-Np responsible for all known signalingactivities. The first step of the autoprocessing reaction, mediated bythe carboxy-terminal sequences within the precursor, entails an internalrearrangement at the site of cleavage to replace the scissile peptidebond by the thioester involving a Cys side chain. In the second stepcholesterol supplies the nucleophile (the 3β-OH) that attacks thethioester intermediate, and remains covalently attached as an adduct toShh-Np (11, 13). Autoprocessing thus is required to release activesignal and the cholesterol adduct restricts the tissue distribution ofthe signal by causing it to associate with the cell surface (12,13).

Given this role of cholesterol in the giogenesis of Hedgehog proteins,an effect of these compounds on Shh-Np production is a particularlyappealing possibility since jervine and cyclopamine structurallyresemble cholesterol (FIG. 1) and AY 9944 and triparanol inhibitspecific late-acting cholesterol biosynthetic enzymes (17, 18, 19, 22).To examine potential effects of these compounds on Shh processing weutilized HK293 cells cultured in lipid-depleted serum and carrying astable integrated construct for expression of Shh underecdysone-inducible control (47). Shh protein expression in these cellscan be induced by addition of muristerone A, an ecdysone analog. Asobserved in embryos this protein is efficiently processed (FIG. 4A,lanes 1 and 2), with little or no detectable accumulation of precursor(M_(r) 45 kD). Addition of jervine, cyclopamine, tomatidine, AY 9944, ortriparanol during the 24-hour induction period did not diminishShh-N_(p) production nor induce accumulation of unprocessed precursor,even at doses 5-fold higher than those required to completely inhibitShh signaling (FIG. 4A, lanes 4-13). All of the amino-terminal cleavageproduct generated in the presence of these compounds is detected in celllysates, not the culture medium (data not shown), and has the sameelectrophoretic mobility as cholesterol-modified Shh-N_(p). Theseobservations are consistent with the presence of a sterol adduct in theamino-terminal cleavage product, since lack of such an adduct isassociated with release into the medium and with decreasedelectrophoretic mobility (the unprocessed amino-terminal fragment isdesignated Shh-N to distinguish it from processed Shh-Np; see lanes 8,9, 17). We also failed to observe any change in efficiency of Shhprocessing or behavior of Shh-NP in transiently-transfected COS-7 or QT6CELLS treated with these compounds (48). We also have observed thatchick embryos treated with jervine after floor plate induction displayedthe normal apical localization of Shh protein within floor plate cells(49).

Because of their structural similarity to cholesterol, we alsoinvestigated the potential effects of the plant compounds on an in vitroautoprocessing reaction utilizing purified components. The proteinutilized in this reaction is derived by replacement of all but sixcodons of the Drosophila Shh amino-terminal coding region with sequencesencoding a hexahistidine purification tag (10). The resulting 29 kDaprotein, His6Hh-C, in purified form undergoes autoprocessing in acholesterol-dependent manner to yield a 25 kD product (50). As seen inFIG. 5A neither jervine, cyclopamine, nor tomatidine inhibit thischolesterol-stimulated autoprocessing reaction, even at concentrations27-fold higher than that of cholesterol. Given the presence of 3β-OH ineach of the plant compounds (FIG. 1), we also tested their ability toreplace cholesterol in providing the nucleophilic group duringprocessing. As seen in FIG. 5B. no appreciable cleavage is stimulated byaddition of these compounds in the absence of cholesterol.

The observation that cholesterol synthesis inhibitors such as AY 9944and triparanol do not inhibit processing raises the possibility thatcholesterol biosynthetic precursors, which accumulate in treated cells(see below), may participate in the reaction. FIG. 5C shows that the invitro reaction can be driven by desmosterol, 7-dehydrocholesterol(7DHC), and lathosterol with efficiencies similar to that ofcholesterol. Desmosterol and 7DHC are the major precursors reported toaccumulate in cells treated with triparanol and AY 9944, respectively.Lanosterl, a 30 carbon cholesterol precursor, on the other hand isunable to participate in the reaction, perhaps due to stericinterference by the two methyl groups attached to the C4 carbon near the3-hydroxyl. In other studies of this in vitro reaction we have observeda requirement for an unhindered hydroxyl at the 3β position on a sterolnucleus, although neither the 8-carbon side chian nor the number orposition(s) of the double bond(s) in the sterol nucleus appear tocritically affect efficiency (51). These observations suggest that all27 carbon sterol intermediates in the biosynthetic pathway are potentialadducts in the autoprocessing reaction, and may account for theunimpaired efficiency of processing in the presence of distal synthesisinhibitors. Thus, although the extent of Shh processing in culturedcells and its localization in vivo appears to be unaffected by theseinhibitory compounds (FIG. 4), we can not rule out the possibility thatthe sterol adduct may differ and that such an abnormally modified signalmay have distinct biological properties.

Inhibitory Compounds Specifically Affect the Response to Shh Signaling

Since our studies of processing provided n evidence for an inhibitoryeffect of these compounds on Shh signal production, we examined thealternative possibility that these compound affect response of targettissues. For these studies we utilized an intermediate neural plateexplant lacking any endogenous source of inducing signal (41, see FIG.6A). Recombinant Shh-N protein (45, 52, 53, 54), lacking a steroladduct, suppresses molecular markers such as Pax7 (55, see FIGS. 6B, C),normally expressed in dorsal cell types, and induces ventral markerssuch as Isl-1 and HNF3β (FIGS. 6D,E), normally expressed in motorneurons and floor plate cells. These cellular responses are elicited ina concentration-dependent manner, with repression of Pax7 observed atconcentrations of Shh-N that are insufficient for induction of HNF38(ref. 55.2 nM. FIGS. 6B,C). Isl-1 and HNF3β occurring at the expense ofIsl-1 (note that the induction of Isl-1 at 6.25 nM Shh-N in FIG. 6D isabolished at 25 nM in 6E).

The teratogenic compounds are able to block completely the repression ofPax7 (at 2 nM Shh-N, FIGS. 6F-I) and the induction of Isl-1 and HNF3β(at 25 nM Shh-N; FIG. 6)-S). In addition tomatidine produces partialinhibition, but only at concentrations 100200 fold higher than thoserequired for complete inhibition by jervine and cyclopamine (FIG. 6T). Acomplete inhibition of the 24 nM response to Shh-N requires does ofteratogenic compounds 2-4 higher than those required to completely blockthe 2 nM response; inhibition of responses to higher concentrations ofShh-N requires higher drug concentrations. Another dose dependent effectcan be noted in FIGS. 6K-N, where drug concentrations two fold below thethresholds required for complete inhibition of the 25 nM response(induction of HNF3β) result in retention or expansion of Isl-1expression. A similar expansion of Isl-1 at intermediate drugconcentrations was seen for midline explants (FIGS. 3D-G), indicatingthat at a fixed level of stimulation by Shh-N, distinct degrees ofpathway activation can be produced by distinct inhibitor concentrations.

To further examine the specificity of these compounds we tested theireffects on induction of a neural crest-like phenotype by BMP7. The BMP7signaling protein is expressed in ectodermal cells adjacent to theneural plate, and appears to function in induction of neural crest anddorsal neural tube cell fates 956). To avoid contamination withendogenous lateral signals, the explants used for these studies weretaken from the ventral neural plate, but excluded the notochord and themidline (FIG. 7A). Addition of BMP7 protein induced formation ofmigratory cells that express the HNK-1 surface antigen (compare FIGS.7B,C), features characteristic of neutral crest cells (56). Neither cellmigration nor expression HNK-1 were blocked by addition of jervine at 10μM (FIG. 7D), a concentration exceeding that required for a completeblock of Shh-N signaling. Similar results were obtained with tomatidineand with cyclopamine. These compounds also failed to inhibit formationof migratory HNK-1 positive cells from explants containing dorsal neuralplate and contiguous epidermal ectoderm (49), which serves as anendogenous source of BMP activity (56).

Drug Effects upon Cholesterol Homeostatis

Pervious reports indicate that triparanol and AY 944 cause theaccumulation of cholesterol precursors (predominantly desmosterol and7-dehycholesterol (7DI-IC) by specifically inhibiting late-actingenzymes of cholesterol biosynthesis (desmosterol Δ24-reductase and 7DHCΔ7-reductase, respectively, 17, 18, 19, 22), and a preliminary analysisof jervine also revealed an effect upon cholesterol biosynthesis (30). Adirect comparison of the effects of these compounds on human primarylymphoblast cultures (57) revealed that all of them, includingtomatidine, cause a relative decrease in cholesterol levels and anincrease in the levels of other sterols (Table I, ref 58).

Table 1. Teratogenic compounds disrupt cholesterol homeostasis incultured cells. Cholesterol biosynthesis is inhibited in primary humanlymphoblasts cultured in the presence of the teratogenic compounds andtomatidine (58). The sterol profiles (57) from these cultures reveal theaccumulation of multiple 27-, 28- and 29-carbon sterol precursors ofcholesterol (59, 60). Esterification of PM-labeled [3H]-cholesterol inrat hepatoma cells is also inhibited by all of the compounds (63).

TABLE 1 Effects of synthetic and plant-derived compounds on cholesterolhomeostasis. AY9944 Triparanol Jervine Cyclopamine Tomatidine (μM) (μM)(μM) (μM) (μM) Control 0.25 0.5 1.0 0.25 0.5 1.0 1.25 2.5 5.0 1.25 2.55.0 1.25 2.5 5.0 A. Cholesterol Biosynthesis Assay Total Sterols (μg/mgprotein) 9.6 7.6 8.3 8.3 4.1 5.1 3.1 8.8 8.4 10 8.4 8.5 8.3 7.8 7.8 5.6Percent Sterols Cholesterol 95 30 33 34 56 45 51 90 90 88 87 76 68 54 4232 Non-Cholesterol Sterols 1. C27 Sterols a. Desmosterol 1.9 9.1 8.7 6.72.5 2.4 2.7 4.2 7.1 11 b. 7 Dehydrodesmosterol 3.5 3.0 1.9 6.0 4.1 2.90.8 0.8 0.8 0.5 c. Cholesta-7,24-dien- 1.8 1.9 1.6 3.1 2.4 2.6 0.5 0.50.6 0.9 1.6 0.9 0.9 0.7 3β-ol d. Zymosterol 9.3 27 23 1.7 2.0 2.3 2.34.5 4.7 e. Cholesta-8(14)-en-3β-ol 9.7 14 20 9.1 8.7 7.3 1.0 1.7 2.3 0.92.5 2.7 6.7 8.9 8.7 f. 7 Dehydrocholesterol 50 36 16 1.5 1.4 1.3 2.4 4.26.3 19 14 9.8 g. Lathosterol 1.3 6.2 7.3 7.9 4.9 4.7 3.6 h. C27 Sterol 1(mw 384) 5.3 7.0 13 20 i. C27 Sterol 2 (mw 382) 6.0 4.1 4.7 j. C27Oxysterol 1 (mw 400) 1.0 2.4 4.5 k. C27 Oxysterol 2 (mw 400) 2.0 5.0 112. C28 Sterols 0.7 1.1 2.6 4.4 2.6 1.5 1.2 0.7 0.7 1.2 1.3 1.8 1.6 3.C29 Sterols  1 3.3 4.6 7.8 8.1 5.5 0.8 0.8 1.6 0.7 1.2 3.1 3.1 AY9944Triparanol Jervine Cyclopamine Tomatidine Percent Inhibition(incorporation (μM) (μM) (μM) (μM) (μM) of label into cholesterylester*) 2.5 5.0 10 2.5 5.0 10 2.5 5.0 10 2.5 5.0 10 2.5 5.0 10 B.Cholesterol Esterification Assay ³H-Cholesterol 39 56 68 49 57 79 24 4450 48 67 79 31 33 39 ¹⁴C-Oleic Acid 20 35 51 54 65 81 28 36 49 45 62 7430 64 52 *The percent of label taken up that was converted tocholesteryl ester was 8%/hour for ³H-cholesterol and 3.6%/hour for¹⁴C-oleic acid

The accumulating sterols largely comprise established intermediates inthe cholesterol biosynthetic pathway or closely related species thatmight be generated by action of the giosynthetic enzymes upon theseintermediates (59). Tomatidine would appear to be the exception to thisgeneral rule, with accumulation to relatively high levels of severalunusual sterols (60).

Reduction of cholesterol levels coupled with an accumulation ofcholesterol biosynthetic precursors are effects observed for a group ofcompounds that have been termed class 2 inhibitors of cholesterolbiosynthesis (61, 62). These compounds appear to act by inhibitingsterol flux between the plasma membrane (PM) and the endoplasmicreticulum (ER). Since cholesterol biosynthetic enzymes are located inthe ER. and sterol precursors of cholesterol are highly concentrated inthe PM, such a block in transport results in an overall reduction ofcholesterol levels. We measured the effects of the synthetic and plantcompounds upon esterification of exogenously added 3H-labelledcholesterol (63) a process which requires transport of PM cholesterol tothe ER. We observed inhibition of esterification oat levels ranging for25-75% for these compounds. An effect of AY 9944 on sterol transportpreviously has been reported (23), but this is the least active of thecompounds we tested in inhibition of esterification. Our data thereforesuggest that transport inhibition may be a factor in the effects of allof these compounds on sterol profiles, consistent with the generalaccumulation of multiple cholesterol biosynthetic precursors. Inaddition however, AY 9944 and triparanol cause accumulation to highlevels of 7DHC and desmosterol, respectively consistent with thewell-known effects of these compounds on the 7DHC A7-reductase anddesmosteroal Δ24-reductase enzymes.

Discussion

The teratogenic effects of distal inhibitors of cholesterol biosynthesishave been known and studied for more than thirty years (14, 15).Similarly, cyclopamine and jervine were identified about thirty yearsago as the plant compounds responsible for the teratogenic effects ofthe range plant Veratrum californicum (28, 29). The most dramaticteratogenic effect of these compounds is the induction of cyclopia andother features of severe holoprosencephaly (HPE); the recent discoverythat HPE is also caused by mutations at the murine and human locisuggested the possibility that these compounds may act to block the Shhsignaling pathway. Our studies have verified the HPE-inducing propertiesof these compounds in chick embryos. We have further examined the earlymolecular correlates of these teratogenic effects and have demonstratedthat these compounds block the induction by Shh protein of ventral celltypes in chick neural plate explants.

Despite the inhibitory effects of these teratogens on cholesterolbiosynthesis (17, 18, 19, 22, 30, see above), we found that none of thecompounds appears to interfere with Shh processing in cultured cells,and that the plant alkaloids neither participate in nor inhibit an invitro Hh protein autoprocessing reaction utilizing purified components.Instead, it is the response to Shh signaling that is affected, asindicated by failure of exogenously added Shh-N to induce ventral celltypes in the presence of teratogenic compounds. Furthermore, althoughexogenously added Shh-N protein can induce endogenous Shh geneexpression in neural plate explants (64, 65), we have demonstrated acomplete inhibition of response by these teratogens at 2 nM Shh-N, aconcentration at which there is not induction of floor plate cells andtherefore no endogenous Shh expression. The inhibitory effects of thesecompounds are dose-dependent, as demonstrated: (1) by maintenance oreven expansion of the Isl-1 intermediate fate at intermediate inhibitorconcentrations below those required for complete inhibition; and (2), bythe requirement for correspondingly higher concentrations of teratogeniccompounds to inhibit the response to increasing levels of Shh-N protein.A further indication of the specificity of these effects is theinability of these compounds to block cell behaviors such as migrationexpression of Pax7, or HNK-1, or the response to other inductive signalssuch as BMP7 at concentrations that completely block the response to Shhsignaling.

Our studies of sterol synthesis and transport suggest that thesecompounds are acting as class 2 inhibitors of cholesterol biosynthesis(61). For several reasons, however, simple reduction of cholesterollevels seem unlikely to account for the effects of these compounds onShh signaling. First, the non-teratogenic compound tomatidine alsodisplays potent inhibitory effects on cholesterol synthesis. Second Shhsignaling in explants is not inhibited by 25-hydroxycholesterol, ahydroxysterol that blocks de novo cholesterol biosynthesis (66). We canalso rule out an inhibitory role for specific sterol precursors that mayaccumulate in drug-treated cells, since addition of 25-hydroycholsteroltogether with inhibitory compounds should eliminate synthesis of sterolprecursors yet does not restore the ability to respond to Shh signaling(67). An alternative mechanism to simple reduction of cholesterol wouldbe a disruption of intracellular transport.

We have also shown that triparanol, jervine, and cyclopamine are potentinhibitors of PM cholesterol esterification, consistent with theirclassification as class 2 inhibitors. Consistent with transportdisruption as the mechanism of drug action in inhibiting Shh signaling,we have found that several other previously characterized class 2compounds also are able to inhibit the response to Shh signaling inexplants (68). Tomatidine, however, also blocks esterification,indicating that general inhibition of this transport pathway is notsufficient for an inhibitory effect on the Shh response. We arecurrently investigating the possibility that this pathway comprisesmultiple steps that are differentially affected by tomatidine and theteratogenic compounds, and that only those steps not essential for theShh response are affected by tomatidine. The unusual sterols thataccumulate in tomatidine-treated cells are associated with peroxisomalsterol metabolism (60), consistent with such a differential effect oftomatidine on intracellular sterol transport.

In light of these drug effects on cholesterol homeostatis, it isinteresting to note the presence of a sterol sensing domain (SSD) withinPtc, a key regulator of the Shh signaling pathway (33). The Ptc SSDinitially was detected as a region of similarity to the Niemann-Pick C.Disease (NP-C) gene (31, 32). The similarity between Ptc and the NP-Cprotein extends beyond the five transmembrane spans of the SSD toinclude all twelve of the proposed transmembrane spans of Ptc. Thesignificance of this sequence homology is not known, and the role of theSSD in NP-C is not clear, although this protein is proposed to regulateintracellular trafficking and loss of its function leads to lysosomalcholesterol accumulation (69). The SSDs of other proteins conferdifferential responses to high and low levels of intracellular sterols.The HMGCoA reductase enzyme thus displays a 3-5 fold decrease instability as sterol concentration rise, and this behavior is dependenton the presence of the SSD. The SCAP regulator protein at low (but notat high) sterol concentrations stimulates the activity of the S2Pmetalloprotease, resulting in cleaveage and activation of the SREBPtranscription factor.

Those of the class 2 cholesterol synthesis inhibitors which have beenexamined appear to increase HMGCoA reductase activity and to stimulatethe cleaveage of SREBP. Given the localization of these two proteins tothe ER, a likely mechanism for this effect is that disruption of steroltransport from PM to ER by class 2 compounds induces a low sterol statein these ER proteins, despite higher levels of cellular sterols overall.The teratogenic compounds studied here all affect cholesterol synthesisand transport, and it is conceivable that they alter the normaldistributions of sterols within intracellular compartments If thefunction of Ptc is critically dependent upon the sterol concentrationsin particular compartment, skewed sterol distributions in thiscompartment could act to perturb Ptc function via its SSD. One otherpossibility is that the function of Ptc in Shh signaling involvesregulation of intracellular transport, as has been suggested for therelated NP-C protein. If this were true, the perturbations of transportgenerated by these teratogenic compounds might affect the transportfunctions of Ptc in such a manner as to inhibit Shh signaling.

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41. Hamburger and Hamilton stage 9-10 (8-10 somites) embryos were usedfor all explant assays. Dissections were carried out in Leibovitz's L15medium (Gibco BRL). Midline tissue just rostral to Hensen's node andwell caudal to the last somite was removed with fine scissors. Theneural ectoderm was separated from the lateral plate mesoderm andendoderm with dispase (Boehringer Mannheim, grade II 2.4 U/ml) treatmentand then washed in L15. Midline, intermediate and ventral neural plateexplants were further dissected with tungsten needles as diagrammed inFIGS. 3A, 6A and 7A. Dissected tissues were transferred to a chamberedcoverglass (Nunc) in a drop of collagen (vitrogen 100, CollagenBiomaterials. Palo Alto, CA) containing 1× modified Eagle's medium(Gibco BRL) and 24 mM NaH₂CO₃ (final pH 7.4-7.6), and warmed to 37.5° C.for 30 minutes (in the absence of CO₂) for gelation. Explants werecultured in 400 μl of F12 Nutrient Mixture (Ham) with glutamine (GibcoBRL), containing N-2 supplement (1×, Gibco BRL) and 100 U/ml penicillinand 100 ug/ml streptomycin in a 5% CO₂, humidified incubator at 37° C.AY 9944, triparanol, jervine, cyclopamine and tomatidine (all from 10 mMstocks in 95% ethanol, except AY 9944 which is water soluble), purifiedShh-N and BMP 7 were added at the initiation of the cultures. All of theexplants were cultured for 40-48 hours except for the intermediateneural plate explants assayed for pax7 repression, which were culturedfor 20-22 hours. At the end of the incubation period, explants werefixed in 4.0% formaldehyde (EM grade, Polysciences, Inc.) in PBS for 1hour at 4° C., washed with PBS and then stained with a secondaryantibody for 2 hours at room temperature. Rabbit anti-rat HNF3β (K2)1:2000, mouse anti-ISL1 (40.2D6) 1:1000, mouse anti-pax7 1:10, mouseanti-rat HNK-1/N-CAM (sigma Biosciences) 1:1000, FITC-conjugated donkeyanti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.) 1:100 andLRSC-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearchLaboratories, Inc.) 1:300 were all diluted in PBTS. The explants wereexamined with an Olympus IX60 inverted microscope using a planapoobjective with a 1.4 numerical aperture. Images were generated byconfocal laser scanning microscopy with a cripton-argon laser excitingat 488 and 568 nm with emissions at 450-550 and 550-650 nm and utilizingOz with Intervission software (Noran) on a Silicon Graphics Inc.platform.

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47. HK293 cells, stably transfected with Shh using theEcdysonc-Inducible Mammalian Expression System (invitrogen). were platedin 6-well culture plates (Flacon, well area 9.6 cm²) in Dulbecco'smodified Eagle's medium (DMEM, Gibco). 10% fetal bovine serum (FBS), 400μg/ml Zeocin Invitrogen), 2 mM L-glutamine, 100 U/ml Penecillin, 100μg/ml Stregtomycin, 350 μg/ml G418 (Invitrogen) at 30-40% confluency andgrown at 37° C. The following day, the media was changed to one thatcontained 10% dilapidated serum (K. M. Gibson et al, J. Lipid Res. 31,515 (1990)) and 1% ITS (Sigma) and otherwise was the same as above.After 24 hours, the cells were induced to express Shh with the additionof 1 μM muristerone A (Invitrogen). AY 9944, triparanol, jervine,cyclopamine and tomatidine (all from 10 mM stocks in 95% ethanol, exceptAY 9944 which is water soluble) were added to the cultures at the timeof induction. The control cells received 0.475% ethanol to equal themaximum ethanol concentration in the 50 μM steriodal alkaloidtreatments. After an additional 24 hours, the culture supernatants wereremoved and the cells were lysed in the plate with 3× SDS-PAGE celllysis buffer (3% SDS), diluted two-fold with water and boiled. Lysatesamples (and in a separate experiment supernatant samples, for which thedata is not shown) were loaded onto SDS-12% polyacrylamide gels foranalysis, immunoblotted with primary antibodies for Shh-N and actin(Amersham) and horseradish peroxidase-conjugated secondary antibodies(Jackson ImmunoResearch Laboratories, Inc.), and visualized with a withluminescent substrate (Pierce).

48. Shh processing in transiently transfected cells is ineffecient, withaccumulation of 50-80% of Shh protein as unprocessed precursor. Even inthese circumstances, we did not observe any effect of jervine,cyclopamine, or tomatidine upon Shh processing efficiency.

49. Unpublished data.

50. The in vitro studies of Hh autoprocessing used a bateriallyexpressed derivative of the Drosophila Hh protein (Porter 96A). Thereactions were carried out as described (Porter 96B), except that thesterols and steroidal alkaloids were dried down from an ethanol orchloroform stock and resuspended in a 0.2% Triton-X 100 solution in abath sonicator prior to addition to the reaction mixture.

51. Other sterols that participate in the reaction with similarefficiency to cholesterol are β-sitosterol, 5-androsten-3β-ol,ergosterol. 4β-hydroxycholesterol. 19-hydroxycholesterol.20α-hydroxycholesterol, 22(S)-hydroxycholesterol,22(R)-hydroxycholesterol and 25-hydroxycholesterol. Epicholesterol,cholesterol acetate, α-ecdysone, 20-OH ecdysone and thiocholesterol areunable to participatein the reaction.

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54. A. Lopez-Martinez, et al., Current Biology 5, 791-796 (1995).

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57. Pooled human lymphoblasts were washed with serum free RPMI-1640,then plated in 35 mm microwells in RPMI-1640 with 15% delipidated FBS(Gibson 90) and cultured at 37° C. in a 5% CO, humidified atmosphere for12 hours. AY 9944, triparanol, jervine, cyclopamine or tomatidine wasthen added and the cells were incubated for five days, after which theneutral sterols were extracted and analyzed as described by R. I. Kelley(Clin. Chim. Acta 236, 45 (1995)). Briefly, pelleted cells weresaponified at 60° C. in 4% (w/v) KOH in 90% ethanol with epicoprostanolas carrier, mixed with an equal volume of water and extracted threetimes in hexane. The hexane extracts were dried under nitrogen,derivatized with bistrimethylsilyltrifluoroacetamide (BSTFA, Pierce) inpyridine and analyzed by selected ion monitoring gaschromatography/mass-spectrometry (SIM-GC/MS), utilizing a HewlettPackard (HP) 5890A splitless injection port, a 0.2 mm×25 m HP-1methylsilicone (0.33 μm liquid phase) capillary column and a HP 5970Amass selective dector operated in electron impact mode at 70 eV with anion source temperature of 200° C.

58. For determining their effects on sterol composition, AY 9944 andtriparanol were used at 0.5μM and jervine, cyclopamine, and tomatidinewere used at 10 μM. Doses lower than these produced more normal sterolprofiles; higher doses increased the relative levels of cholesterolprecursors but also reduced cell growth during the five day incubationperiod of this assay.

59. Sterols 1a, 1c-g, 2a,b and 3a,b are all intermediates in normalcholesterol biosynthesis, and 1b is thought to derive from 1a (G. Salenet al., J. Lipid Res. 37, 1169 (1996)).

60. Sterol 1h is associated with peroxisomal sterol synthesis and isparticularly prominent in tomatidine treated cells. Sterol 4 is seenonly in normal cells treated with tomatidine, but not intomatidine-treated cells from Zellweger's Syndrome patients, which lackperoxisomes. Sterol 4 is an apparent dihydroxy-ketosterol whosestructure is not yet fully resolved.

61. Y. Lange, T. L. Steck, J. Biol. Chem. 269, 29371-29374 (1994).

62. Y. Lange, T. L. Steck, Trends in Cell Biol. 6, 205-208 (1996).

63. Esterification of plasma membrane [³H] cholesterol in hepatoma cellswas assaved according to Lange and Steck. Briefly, AH22 Hepatoma cellswere cultured in 25 cm² flasks to ˜89-90% confluency in DMEM 10% FBS at37° C. The cells were washed in PBS and then labeled with 1.38 μCi [³H]cholesterol (3.17×10⁻⁵ mmol cholesterol) in PBS for 10 minutes at 37° C.The [³H]cholesterol was in a vortexed solution of 2.5% Triton WR-1339,2.5 mM NaPi (pH 7.5) and 0.125 M sucrose. The cells were then washed inPBS with 0.5 mg/ml bovne serumalbumin (BSA) and incubated for 1.5 hoursat 37° C. in DMEM 10%FBS without or with AY 9944, triparanol, jervine,cyclopamine or tomatidine. The cells were detached with trypsin, washedand suspended in 1 ml PBS. The sterols were then extracted with 2.5 mlof chloroform:methanol (2:1), dried on a speed vacuum concentrator,resuspendedin 50 μl of chloroform and spotte don solica gel G coated TLCplates (Merck). Cholesteryl esters and cholesterol were fractionatedwith a heptane:ether:acetic acid solvent (20:5:1), dried, visualizedwith I₂ vapor, scraped and counted directly in an aqueous scintilationcounting cocktail (Econo-Safe, Research Products International Corp.)

64. E. Marti, D. A. Bumcrot, R. Takada, A. P. McMahon, Nature 375,32)2325 (1995).

65. Thomas M. Jessell, personal communication.

66. None of the explant responses to treatment with 2 nM or 25 nM Shh-Nwere affected by additional of 25-OH cholesterol at 25 μM. 25-OHcholesterol is a potent inhibitor of HMG CoA reductase and at theconcentrations used blocks de novo cholesterol synthesis in chickembryos and in cultured cell systems (data not shown; S. C. Miller andG. Melnykovych, J. Lipid Res. 25, 991 (1984); J. J. Bell, T. E. Sargeantand J. A. Watson, J. Bio. Chem. 251, 1745 (1976)).

67. Addition of 25 μM 25-hydroxycholesterol to explant cultures did notreverse the inhibitory effects of any of the teratogenic compounds.

68. Class 2 cholesterol synthesis inhibitors at the given concentrationsblock the response of intermediate neural plate explants to 25 nM Shh-N,without affecting signaling by BMP7: U 18666A 0.25 μM, chloroquine 50μM, imipramine 75 μM, progesterone 20 μM.

69. P. G. Pentchev, et al. Biochimica et Biophysica Acta 1225, 235-243(1994).

All of the references cited above are hereby incorporated by referenceherein.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method for inhibiting activation of a hedgehog-patched pathway in a patient diagnosed with a hyperproliferative disorder, comprising administering to the patient a composition comprising a purified hedgehog antagonist in a sufficient amount to reduce the activation of the hedgehog-patched pathway in a cell of the patient, wherein the antagonist is a steroidal alkaloid having a structure represented in the general formula (I), or unsaturated forms thereof and/or nor- or homo-derivatives thereof:

wherein, as valence permits, R₂, R₃, R₄, and R₅, represent one or more substitutions to the ring to which each is attached, for each occurrence, independently represent hydrogen, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, =O, =S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH₂)_(m)—R₈; R₆, R₇, and R′₇, independently for each occurrence, are absent or represent hydrogens, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, =O, =S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH₂)_(m)—R₈, or R₆ and R₇, or R₇ and R′₇, taken together form a ring or polycyclic ring, with the proviso that at least one of R₆, R₇, or R′₇ is present and includes a primary or secondary amine; R₈ represents an aryl, a cycloalkyl, a cycloaklenyl, a heterocycle, or a polycycle; and m is an integer in the range 0 to 8 inclusive.
 2. The method of claim 1, wherein: R₂ and R₃, for each occurrence, is an —OH, alkyl, —O—alkyl, —C(O)—alkyl, or —C(O)—R₈; R₄, for each occurrence represents H, —OH, =O, alkyl, —O—alkyl, —C(O)—alkyl, or —C(O)—R₈; R₆, R₇, and R′₇ each independently represent , hydrogen, alkyls, alkenyls, alkynyls, amines, imines, amides, carbonyls, carboxyls, carboxamides, ethers, thioethers, esters, or —(CH₂)_(m)—R₈, or R₇, and R′₇ taken together form a furanopiperidine, such as perhydrofuro[3,2-b]pyridine, a pyranopiperidine, a quinoline, an indole, a pyranopyrrole, a naphthyriding, a thiofuranopiperidine, or a thiopyranopiperidine with the proviso that at least one of R₆, R₇, or R′₇ is present and includes a primary or secondary amine: R₈ represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle, or a polycycle, and preferably R₈ is a piperidine, pyrimidine, morpholine, thiomorpholine, pyridazine.
 3. A method for inhibiting activation of a hedgehog-patched pathway in a patient diagnosed with a hyperproliferative disorder, comprising administering to the patient a composition comprising a purified hedgehog antagonist in a sufficient amount to reduce the activation of the hedgehog-patched pathway in a cell of the patient, wherein the antagonist is a steroidal alkaloid having a structure represented in the general formula (II), or unsaturated forms thereof and/of nor- or homo-derivatives thereof:

wherein, as valence permits, R₂, R₃, R₄, and R₅, represent one or more substitutions to the ring to which each is attached, for each occurrence, independently represent hydrogen, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, =O, =S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH₂)_(m)—R₈; R₆, R₇, and R′₇, are absent or represent, independently, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, =O, =S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, etheres, thioethers, alkylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH₂)_(m)—R₈, or R₆ and R₇, or R₇ and R′₇, taken togehter form a ring or polycyclic ring, with the proviso that at least one of R₆, R₇, or R′₇ is present and includes a primary or secondary amine; R₈ represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle, or a polycycle; and m is an integer in the range 0 to 8 inclusive; and X represents O or S.
 4. A method for inhibiting activation of a hedgehog-patched pathway in a patient diagnosed with a hyperproliferative disorder, comprising administering to the patient a composition comprising a purified hedgehog antagonist in a sufficient amount to reduce the activation of the hedgehog-patched pathway in a cell of the patient, wherein the antagonist has a structure represented in the general formula (III), or unsaturated forms thereof and/or nor- or homo-derivatives thereof;

wherein, as valence permits, R₂, R₃, R₄, and R₅, represent one or more substitutions to the ring to which each is attached, for each occurrence, independently represent hydrogen, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, =O, =S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH₂)_(m)—R₈; R₈ represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle, or a polycycle; and A and B represent monocyclic or polycyclic groups; T represents an alkyl, an aminoalkyl, a carboxyl, an ester, an amide, ether or amine linkage of 1-10 bond lengths; T′ is absent, or represents an alkyl, an aminolkyl, a carboxyl, an ester, an amide, ether of amine linkage of 1-3 bond lengths, wheren if T and T′ are present together, than T and T′ taken together with the ring B form a covalently closed ring of 5-8 ring atoms; R₉ represents one or more substitutions to the ring A or B, which for each occurrence, independently represent halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, =O, =S, alkoxyl silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH₂)_(m)—R₈; and n and m are, independently, zero, 1 or 2; with the proviso that A and R₉, or T, T′ B and R₉, taken together include at least one primary or secondary amine.
 5. A method for inhibiting activation of a hedgehog-patched pathway in a patient diagnosed with a hyperproliferative disorder, comprising administering to the patient a composition comprising a purified hedgehog antagonist in a sufficient amount to reduce the activation of the hedgehog-patched pathway in a cell of the patient, wherein the antagonist has a structure represented in the general formula (IV), or unsaturated forms thereof and/or nor- or homo-derivatives thereof:

wherein, as valence permits, R₂, R₃, R₄, And R₅, represent one or more substitutions to the ring to which each is attached, for each occurrence, independently represent hydrogen, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, =O, =S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH₂)_(m)—R₈; R₆ is absent or represents halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, =O, =S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arysulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH₂)_(m)—R₈; R₈ represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle, or a polycycle; R₉ represents one or more substitutions to the ring A or B, which for each occurrence, independently represent halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, =O, =S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH₂)_(m)—R₈; m is 0, 1, or 2; and R₂₂ is absent or represents an alkyl, an alkoxyl or —OH.
 6. A method for inhibiting activation of a hedgehog-patched pathway in a patient diagnosed with a hyperproliferative disorder, comprising administering to the patient a composition comprising a purified hedgehog antagonist in a sufficient amount to reduce the activation of the hedgehog-patched pathway in a cell of the patient, wherein the antagonist has a structure represented in the general formula (V) or unsaturated forms thereof and/or nor- or homo-derivatives thereof:

wherein, as valence permits, R₂, R₃, R₄, and R₅, represent one or more substitutions to the ring to which each is attached, for each occurrence, independently represent hydrogen, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, =O, =S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH₂)_(m)—R₈; R₆ is absent or represents halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, =O, =S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers thioethers, alkylsulfonyls, arylsulfonyls, selesnoethers, ketones, aldehydes, esters, or —(CH₂)_(m)—R₈; R₈ represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle, or a polycycle; R₉ represents one or more substitutions to the ring A or B, which for each occurrence, independently represent halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl, =O, =S, alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls, phosphonates, phosphines, carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers, alkylsulfonyls, arysulfonyls, selenoethers, ketones, aldehydes, esters, or —(CH₂)_(m)—R₈; and m is 0, 1, or
 2. 7. The method of claims 1, 3, 4, 5, or 6, wherein the hedgehog antagonist does not substantially interfere with the biological activity of aldosterone, androstane, androstene, androstenedione, androsterone, cholecalciferol, cholestane, cholic acid, corticosterone, cortisol, cortisol acetate, cortisone, cortisone acetate, deoxycorticosterone, digitoxigenen, ergocalciferol, ergosterol, estradiol-17α, estradiol-17-β, estriol, estrane, estrone, hydrocortisone, lanosterol, lithocholic acid, mestranol, β-methasone, prednisone, pregnane, pregnenolone, progesterone, spironolactone, testosterone, or triamcionolone.
 8. The method of claim 1, 3, 4, 5, or 6, wherein the hedgehog antagonist does not specifically bind a nuclear hormone receptor.
 9. The method of claim 1, 3, 4, 5, or 6, wherein the hedgehog antagonist does not specifically bind estrogen or testosterone receptors.
 10. The method of claim 1, 3, 4, 5, or 6, wherein the hedgehog antagonist has no estrogenic activity at therapeutic concentrations.
 11. The method of claim 1, 3, 4, 5, or 6, wherein the hedgehog antagonist inhibits activation of the hedgehog-patched pathway with an ED₅₀ of 1 mM or less.
 12. The method of claim 1, 3, 4, 5, or 6, wherein the hedgehog antagonist inhibits activation of the hedgehog-patched pathway with an ED₅₀ of 1μM or less.
 13. The method of claim 1, 3, 4, 5, or 6, wherein the hedgehog antagonist inhibits activation of the hedgehog-patched pathway with an ED₅₀ of 1 nM or less.
 14. The method of claim 1, 3, 4, 5, ro 6, wherein the hedgehog antagonist is administered as part of a therapeutic or cosmetic application.
 15. The method of claim 1, 3, 4, 5, or 6, wherein the hyperproliferative disorder comprises basal cell carcinoma.
 16. The method of claim 1, 3, 4, 5, or 6, wherein the hyperproliferative disorder comprises medulloblastoma.
 17. The method of claim 1, 3, 4, 5, or 6, wherein the composition is administered topically.
 18. The method of claim 1, 3, 4, 5, or 6, wherein the antagonist is other than jervine. 